Potent LNA oligonucleotides for the inhibition of HIF-1a expression

ABSTRACT

The present disclosure relates to an LNA oligonucleotide consisting of a sequence selected from the group consisting of 5′-( T   x )G x G x c s a s a s g s c s a s t s c s c s T x G x   T -3′ and 5′-( G   x )T x T x a s c s t s g s c s c s t s t s c s T x T x   A -3′, wherein capital letters designate a beta-D-oxy-LNA nucleotide analogue, small letters designate a 2-deoxynucleotide, underline designates either a beta-D-oxy-LNA nucleotide analogue or a 2-deoxynucleotide, subscript “s” designates a phosphorothioate link between neighbouring nucleotides/LNA nucleotide analogues, and subscript “x” designates either a phosphorothioate link or a phosphorodiester link between neighbouring nucleotides/LNA nucleotide analogues, and wherein the sequence is optionally extended by up to five 2-deoxynucleotide units. The LNA oligonucleotides are useful for modulating the expression of hypoxia-inducible factor-1a (HIF-1a), e.g. in the treatment of cancer diseases, inhibiting angiogenesis, inducing apoptosis, preventing cellular proliferation, or treating an angiogenic disease, e.g. diabetic retinopathy, macular degeneration (ARMD), psoriasis, rheumatoid arthritis and other inflammatory diseases.

This application is a divisional of U.S. patent application Ser. No.11/271,686, filed on Nov. 9, 2005, which claims the benefit of U.S.Provisional Patent Application No. 60/626,563, filed Nov. 9, 2004; U.S.Provisional Patent Application No. 60/647,186, filed Jan. 25, 2005; U.S.Provisional Patent Application No. 60/699,721, filed Jul. 15, 2005; andU.S. Provisional Patent Application No. 60/724,621, filed Oct. 7, 2005,the entire teachings of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of HIF-1a. In particular, this invention relates to LNAoligonucleotides, which are specifically hybridisable with nucleic acidsencoding HIF-1a. The LNA oligonucleotides have been shown to modulatethe expression of HIF-1a and pharmaceutical preparations thereof andtheir use as treatment of cancer diseases, inflammatory diseases and eyediseases are disclosed.

BACKGROUND OF THE INVENTION

Solid tumors must establish a blood supply and have enhanced glucosemetabolism to grow beyond a few millimeters. How they sense hypoxia, andrespond by activating hypoxia-inducible genes and secreting angiogenicfactors to establish a blood system is central to cancer biology. Manytumors contain hypoxic microenvironments, which have been associatedwith malignant progression, metastasis and resistance to radiotherapyand chemotherapy.

The discovery of hypoxia-inducible factor-1 (HIF-1) gave some insightinto the regulation of hypoxia-inducible genes (U.S. Pat. No. 5,882,914and WO 96/39426; WO 99/48916). HIF-1 is composed of two subunits HIF-1α(HIF-1alpha; referred to herein as “HIF-1a”) and HIF-1β and it binds—1hypoxia-response elements (HREs) in enhancers of genes encodingangiogenic factors such as VEGF and glycolysis-related proteins such asglycolytic enzymes and glucose transporter 1 and 3 (GLU-1 and 3).

It has been demonstrated that engineered down-regulation of HIF-1a byintratumoral gene transfer of an antisense HIF-1a plasmid leads to thedown-regulation of VEGA and decreased tumor microvessel density (WO00/76497, Sun X et al, Gene Therapy (2001) 8, 638-645). The plasmidcontained a 320-bp cDNA fragment encoding 5′-end of HIF-1a (nucleotides152-454; Genebank AF003698).

WO 2003/085110 shows LNA antisense oligonucleotides which down-regulateshuman HIF-1a expression. One compound is named CUR813 (SEQ ID NO. 11).

The present invention disclosed LNA oligonucleotides, which are morepotent than CUR813 (SEQ ID NO. 11). Also the specific LNAoligonucleotides, according to the invention, induce apoptosis andinhibit proliferation. Also, the LNA oligonucleotides which have a 100%sequence identity to the mouse HIF-1a down-regulate the HIF-1aexpression in the liver, colon and kidney in mice.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of HIF-1a. In particular, this invention relates to LNAoligonucleotides over 2 specific motifs targeting HIF-1a. These motifsare disclosed as SEQ ID NOS. 3 and 4. Specifically preferred LNAoligonucleotides are SEQ ID NO. 1 and SEQ ID NO. 2. The LNAoligonucleotides of the invention are potent inhibitors of HIF-1α mRNAexpression and protein levels.

More particularly, the present invention provides an LNA oligonucleotideconsisting of a sequence selected from the group consisting of

(SEQ ID NO. 3) 5′-(T_(x))G_(x)G_(x)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(x)G_(x)T-3′ and (SEQ ID NO. 4) 5′-(G_(x))T_(x)T_(x)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(x)T_(x)A-3′wherein capital letters designate a beta-D-oxy-LNA nucleotide analogue,small letters designate a 2-deoxynucleotide, underline designates eithera beta-D-oxy-LNA nucleotide analogue or a 2-deoxynucleotide, subscript“s” designates a phosphorothioate link between neighbouringnucleotides/LNA nucleotide analogues, and subscript “x” designateseither a phosphorothioate link or a phosphorodiester link betweenneighbouring nucleotides/LNA nucleotide analogues, and where thenucleotide units in the bracket, (T _(x)), (T) or (G _(x)), (A),respectively, represent optional units, and

-   wherein the sequence is optionally extended by up to five    2-deoxynucleotide units.

Pharmaceutical compositions comprising the LNA oligonucleotide of theinvention are also provided. Further provided are methods of modulatingthe expression of HIF-1a in cells or tissues comprising contacting saidcells or tissues with one or more of the LNA oligonucleotides orcompositions of the invention. Also disclosed are methods of treating ananimal or a human, suspected of having or being prone to a disease orcondition, associated with expression of HIF-1a by administering atherapeutically or prophylactically effective amount of one or more ofthe LNA oligonucleotides or compositions of the invention. Further,methods of using LNA oligonucleotides for the inhibition of expressionof HIF-1a and for treatment of diseases associated with HIF-1a activityare provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an increased stability of SEQ ID NO. 1 and SEQ ID NO. 5 inrat plasma (NtacSD male, Li-Heparine (Taconic, M&B)) compared to SEQ IDNO. 6. The oligonucleotides were incubated at 20 μM concentrations at37° C. for 0-, 4-, or 24-hours. No degradation fragments of SEQ ID NO. 1can be detected even after 24 hours digestion.

FIG. 1B shows Stability of Full length SEQ ID NO. 1 and SEQ ID NO. 13, aphosphorothioate and iso-sequential to SEQ ID NO. 1, in Rat and Humanserum. Oligonucleotides were added to human or rat serum at a finalconcentration of 20 μM. The figure shows LNA oligonucletide stability upto 1-96 hours in respectively human and rat serum at 37° C. For ratserum, the second last panel in FIG. 1B demonstrates sustained enzymeactivity even after 48 hours and 96 hours. The latter panel function asa negative control demonstrating no degradation of SEQ ID NO. 1 and SEQID NO. 13 when incubated at 37° C. without plasma added.

FIG. 1C shows extremely long stability of SEQ ID NO. 1 in human and ratplasma. The oligonucleotide was incubated in human or rat plasma for1-96 hours and run on a denaturing gel. Following staining with SyBrgold the amount of full length product was measured by using aphosphorimager and plotted against time.

FIG. 2A shows HIF-1a protein down-regulation in LNA oligonucleotidestransfected U373 cells. U373 cell were transfected with 2 or 10 nMcompound or mock transfected, incubated at hypoxia and analysed forHIF-1a protein down-regulation by Western blotting. Tubulin expressionwas analysed as control of equal loading.

FIG. 2B shows HIF-1alfa protein down-regulation following treatment withSEQ ID NO. 1 in U373 glioblastoma cancer cell lines. Pan-actinexpression was analysed as control of equal loading. Cells weretransfected with 0.2, 1 and 10 nM SEQ ID NO. 1 or SEQ ID NO. 10, whichis a 2 bp mm to SEQ ID NO. 1. The lower panel is a quantification of thegel.

FIG. 2C shows down-regulation of HIF-a expression 24 hours followingtreatment with the HIF-1a targeting LNA oligonucleotide, SEQ ID NO. 1,and a LNA containing scrambled control oligonucleotide SEQ ID NO. 8 inU373 cells. The HIF expression is correlated to either GAPDH orBeta-actin and related to an untransfected control (mock). Following RNApurification, mRNA expression is quantified by QPCR.

FIGS. 3A and 3B shows induction of apoptosis measured as a kineticprofile of induced Caspase 3/7 activity following 24-72 hours treatmentwith LNA oligonucleotides in glioblastoma cell line U373 at normoxia orhypoxia. SEQ ID NO. 1 is shown to be a potent inducer of earlyapoptosis.

FIG. 4A: Induction of early-apoptotic cell stage measured by AnnexinV-FITC and PI flow cytometry analysis after 48 hours. The U373 cellstreated with the LNA oligonucleotide SEQ ID NO. 1 were classified asmore “early apoptotic” compared to mock and SEQ ID NO. 12 treated cells.

FIG. 4B: Quantification of induction of early apoptosis in U373 cellsfollowing treatment with SEQ ID NO. 1. Percentage of cells forced intoearly apoptosis 48 hours following treatment of SEQ ID NO. 1 indifferent dosages. U373 cells were transfected with SEQ ID NO. 1 or twodifferent scrambled control oligonucleotides SEQ ID NO. 8 and SEQ ID NO.12. Following harvest and incubation with Annexin V ab and PI, thenumber of cells in early apoptosis was measured by Flow cytometry.

FIGS. 5A and 5B shows compounds transfected glioblastoma cell line U373cells 24-72 hours after transfection and incubation at either hypoxia ornormoxia. SEQ ID NO. 1 is shown to be a potent inhibitor ofproliferation as measured by MTS assay.

FIG. 6A and FIG. 6B show in vivo endogenous liver target down-regulationof two administration regimens using SEQ ID NO. 1. Measuring mRNA levelsof HIF-1α as well as the downstream target VEGF shows that SEQ ID NO. 1is also an effective inhibitor of said target FIG. 6A: ip injectionsdaily in hairy mice for 14 days. FIG. 6B: ip injections twice weekly inhairy mice for 14 days.

FIG. 6C shows in vivo endogenous kidney HIF-1α after down-regulationadministered ip injections daily in hairy mice for 14 days regimens ofSEQ ID NO. 1.

FIG. 7A shows that SEQ ID NO. 1 is a potent inhibitor measured bydown-regulation of in vivo expression of HIF-1a in liver followingadministration of SEQ ID NO. 1. Different thiolated versions of SEQ IDNO. 1 (SEQ ID NO. 5 and SEQ ID NO. 6) and SEQ ID NO. 1 respectively weredosed to hairy mice at 18 or 3.6 mg/kg daily for 14 days and sacrificed.Expression of HIF-1a was measured at mRNA level by QPCR and normalisedto beta-actin as described in M&M.

FIG. 7B shows that SEQ ID NO. 1 is also a potent inhibitor measured bydown-regulation of in vivo expression of HIF-1a in liver followingadministration of SEQ ID NO. 1. Different thiolated versions of SEQ IDNO. 1 (SEQ ID NO. 5 and SEQ ID NO. 6) and SEQ ID NO. 1 respectively weredosed to hairy mice at 50, 10 or 2 mg/kg twice a week for 14 days andsacrificed. Expression of HIF-1a was measured at mRNA level by QPCR andnormalised to beta-actin.

FIG. 7C shows down-regulation of in vivo expression of HIF-1a in kidneyfollowing administration of SEQ ID NO. 1. Different thiolated versionsof SEQ ID NO. 1 (SEQ ID NO. 5 and SEQ ID NO. 6) were dosed to hairy miceat 18 or 3.6 mg/kg daily for 14 days and sacrificed. Expression ofHIF-1a was measured at mRNA level by QPCR and normalised to beta-actin.

FIG. 8A shows superior in vivo efficacy using SEQ ID NO. 1 compared toSEQ ID NO. 11 and SEQ ID NO. 12 (a scrambled control) measured bytumor-weight of U373 tumors from xenograft. SEQ ID NO. 1, SEQ ID NO. 11and SEQ ID NO. 12 were dosed at 50 mg/kg twice a week for one week inU373 xenograft mice implanted at the ovaries. 2 days following the lastdose animals was sacrificed. At sacrifice tumors were weighed and theindividual tumor weight plus the mean tumor weight (red) was calculatedand plotted. A statistical significant difference (P=0.005) was foundbetween the Control group (a scrambled control SEQ ID NO. 12) and themice treated with a SEQ ID NO. 1.

FIG. 8B shows vessel density in U373 tumors from xenograft treated withSEQ ID NO. 1. SEQ ID NO. 1 was dosed at 50 mg/kg twice a week for oneweek in U373 xenograft mice implanted at the ovaries. 2 days followingthe last dose, animals was sacrificed. Vessel-density was calculatedfollowing CD31 staining and related to the total area. A statisticalsignificant difference (P=0.005) was found between the saline group andthe mice treated with a scrambled control (SEQ ID NO. 12).

FIG. 8C shows staining of CD 31 in sections from U373 tumors implantedat the ovaries and treated with SEQ ID NO. 1 as described for FIG. 8B.

FIG. 8D shows HIF-1α expression quantified by real-time PCR andnormalised to GAPDH in U373 tumors implanted at the ovaries and treatedwith SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 12 and PBS as described forFIG. 8B.

FIG. 9A shows in vivo uptake (in μg per gram tissue) plus targetdown-regulation (% inhibition of HIF-1a mRNA expression correlated toβ-actin expression) of hairy mice following one i.v. dose of SEQ ID NO.1 of 25 mg/kg. SEQ ID NO. 1 has a half-life of approximately 46 hours inkidney and 66 hours in the liver. FIG. 9B upper panel shows SEQ ID NO. 1dosed at 50 mg/kg once i.p. in hairy mice. Five animals treated with SEQID NO. 1 at 50 mg/kg were sacrificed following 1, 3, 4, 5 and 8 daysafter treatment and HIF-1a expression was analysed and normalised toBeta-actin. Expression of HIF-1a was measured at mRNA level by QPCR andnormalised to beta-actin as described in example 8. In the lower panelSEQ ID NO. 1 was dosed at 25 or 50 mg/kg once i.v. in hairy mice. Fiveanimals treated with SEQ ID NO. 1 at 25 or 50 mg/kg were sacrificedfollowing 1, 2, 3, 4, 5 and 8 days after treatment and were analysed forfull length SEQ ID NO. 1 by HPLC methods as described in example 13.Data are presented as μg SEQ ID NO. 1/gram tissue.

FIG. 9C shows HIF-1α expression quantified by real-time PCR andnormalised to GAPDH in mouse liver in mice receiving one dose of 50mg/kg i.p. of SEQ ID NO. 1 and SEQ ID NO. 16 and sacrificed at day 1 and10.

FIG. 10A shows duration of action of SEQ ID NO. 1 inhibiting HIF-1aexpression in xenograft mice dosed 25 mg/kg for 7 days and sacrificed 1or 5 days after the last dose.

FIG. 10B shows in vivo liver, skin tumor and kidney uptake offam-labeled version of SEQ ID NO. 1 (SEQ ID NO. 7) at 25 mg/kg/day forseven days and sacrificed 5 days following the last treatment.

FIG. 10C shows target down-regulation (% inhibition of HIF-1a mRNAexpression correlated to GAPDH expression) plus in vivo uptake (in μgper gram tissue) of SEQ ID NO. 7 in the liver of xenograft mice treatedwith 5 mg/kg/day SEQ ID NO.7, scrambled control SEQ ID NO. 20 or salinei.p. on days 7, 10, 13 and 17 after transplantation as described inexample 17.

FIG. 10D shows target down-regulation (% inhibition of HIF-1a mRNAexpression correlated to β-actin expression) after treatment with SEQ IDNO. 7 or scrambled control SEQ ID NO. 20 plus in vivo uptake (in μg pergram tissue) of SEQ ID NO. 7 in mouse colon treated as described inexample 17.

FIG. 10E shows in vivo uptake (in μg per gram tissue) of SEQ ID NO. 7 inxenograft tumors HT29 and PC3 treated as described in example 17.

FIG. 11 shows in vivo endogenous liver target down-regulation of HIF-1aand VEGF mRNA after 5 doses of 30 mg/kg every 3^(rd) day of SEQ ID NO. 1compared to the one mismatch control SEQ ID NO. 9.

FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D shows expression of VEGFA andMMP-2 following treatment with the HIF-1a targeting LNA oligonucleotide,SEQ ID NO. 1, and a scrambled control SEQ ID NO. 8 in U373 cells. Adose-dependent down-regulation in VEGFA and MMP-2 expression (secretion)is observed 48 hours following treatment with SEQ ID NO. 1 or ascrambled control (SEQ ID NO. 8) in U373 cells. The VEGFA (FIGS. 12A,12B and 12C) and MMP-2 (FIGS. 12D and 12E) expression is related to cellnumber and normalized to mock. In FIGS. 12A, 12C and 12D VEGFA and MMP-2expression is measured 48 hours following treatment, whereas in FIGS.12B and 12E secretion of VEGFA and MMP-2 is quantified 24-120 hoursfollowing tranfection.

FIG. 13 shows down-regulation of HIF-1α protein measured by western blotand disruption of tube formation of HUVEC cells treated with SEQ ID NO.1 at 1 and 5 nM compared to SEQ ID NO. 8 and untreated control.

FIG. 14A Whole body radioluminograms showing the distribution ofradioactivity at 5 minutes a), 4 hours b), 24 hours c) and 18 days d)after a single intravenous administration of ³H-labelled SEQ ID NO. 1 infemale pigmented mice.

FIG. 14B shows the distribution of radioactivity at 5 minutes and 7 daysand that a very strong retention of the ³H-labelled SEQ ID NO. 1compound is observed in bone marrow, kidney, liver, lung, skin, spleen,urine, gastric mucosa, lymph node, uvea of the eye and uterus after 7days.

FIG. 15 shows uptake of a FAM-labelled version of SEQ ID NO. 1 (SEQ IDNO. 7) in different cell types within bone marrow, spleen and peripheralblood 1 hour following administration of SEQ ID NO. 7 compared tountreated cells measured by FACS analysis.

FIG. 16A shows HIF-1α expression measured by real-time PCR andnormalised to 18S RNA in the liver and kidney of cynomolgus monkeystreated with 40, 10 and 6 mg/kg SEQ ID NO. 1 twice a week for 4 weeks.FIG. 16B shows uptake of SEQ ID NO. 1 in liver and kidney of cynomolgusmonkeys one day following the last dose or 4 weeks following the lastdose (recovery animals) treated as described above together with data onrecovery animals (R), which were left untreated for 4 weeks after end oftreatment.

DESCRIPTION OF THE INVENTION

The present invention employs particular LNA oligonucleotides, namelyLNA oligonucleotides comprising the sequence SEQ ID NO. 3 and SEQ ID NO.4, for use in modulating the function of nucleic acid molecules encodingHIF-1a. The modulation is ultimately a change in the amount of HIF-1aproduced. In one embodiment, this is accomplished by providing antisenseLNA oligonucleotides, which specifically hybridise with nucleic acidsencoding HIF-1a. The modulation is preferably an inhibition of theexpression of HIF-1a, which leads to a decrease in the number offunctional HIF-1a proteins produced.

The LNA Oligonucleotides

More particular, the present invention provides an LNA oligonucleotideconsisting of a sequence selected from the group consisting of

(SEQ ID NO. 3) 5′-(T_(x))G_(x)G_(x)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(x)G_(x)(T)-3′and (SEQ ID NO. 4) 5′-(G_(x))T_(x)T_(x)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(x)T_(x)(A)-3′wherein capital letters designate a beta-D-oxy-LNA nucleotide analogue,small letters designate a 2-deoxynucleotide, underline designates eithera beta-D-oxy-LNA nucleotide analogue or a 2-deoxynucleotide, subscript“s” designates a phosphorothioate link between neighbouringnucleotides/LNA nucleotide analogues, and subscript “x” designateseither a phosphorothioate link or a phosphorodiester link betweenneighbouring nucleotides/LNA nucleotide analogues, and where thenucleotide units in the bracket, (T _(x)), (T), or (G _(x)), (A),respectively, represent optional units, and

-   wherein the sequence is optionally extended by up to five    2-deoxynucleotide units.

The terms “LNA oligonucleotide defined herein”, “LNA oligonucleotideaccording to the invention”, and the like, refer to the “LNAoligonucleotide” defined above as well as the embodiments, variants,salts, prodrugs, etc. provided in the following.

The above-defined LNA oligonucleotides based on SEQ ID NO. 3 and SEQ IDNO. 4 have a length of 13-20 nucleotide units. The minimal sequencelength of 13 is obtained if the nucleotide units in the bracket, (T_(x)), (T) or (G _(x)), (A), respectively, are absent, and the maximumsequence length of 20 is obtained if the nucleotide units in thebracket, (T _(x)), (T) or (G _(x)), (A), respectively, are present andif the sequence SEQ ID NO. 3 or SEQ ID NO. 4 is extended by five2-deoxynucleotide units.

In one embodiment, the nucleotide units in the bracket, (T _(x)), (T) or(G _(x)), (A), respectively, are absent, and in another currently morepreferred embodiment, the nucleotide unit in the bracket, (T _(x)), (T)or (G _(x)), (A), respectively, are present. Also interesting are theembodiments, where the 5′-terminal optional unit, (T _(x)) or (G _(x)),respectively, is present and where the 3′-terminal optional unit, (T) or(A), respectively, is absent, and the embodiments where the 5′-terminaloptional unit, (T _(x)) or (G _(x)), respectively, is absent and wherethe 3′-terminal optional unit, (T) or (A), respectively, is present.

The selection of a beta-D-oxy-LNA nucleotide analogue or a2-deoxynucleotide for the underlined nucleotide units in the above SEQID NO. 3 and SEQ ID NO. 4 appears to be less critical. However, in oneembodiment, both of the underlined nucleotide units designate a2-deoxynucleotide. In another currently more preferred embodiment, oneor both of the underlined nucleotide units designate a beta-D-oxy-LNAnucleotide analogue.

In one variant, the 5′-terminal nucleotide unit in the bracket, (T _(x))or (G _(x)), respectively, is absent, and the 3′-terminal otherunderlined nucleotide unit, (T) or (A), respectively, designates a2-deoxynucleotide, or more preferable, a beta-D-oxy-LNA nucleotideanalogue.

In another variant, the 5′-terminal nucleotide unit in the bracket, (T_(x)) or (G _(x)), respectively, designate a 2-deoxynucleotide, or, morepreferable, a beta-D-oxy-LNA nucleotide analogue, and the 3′-terminalother underlined nucleotide unit, (T) or (A), respectively, is absent.

In another variant, the nucleotide units in the bracket are present, andone or both of the underlined nucleotide units designate abeta-D-oxy-LNA nucleotide analogue, i.e. (i) the 5′-terminal underlinednucleotide designates a beta-D-oxy-LNA nucleotide analogue and the3′-terminal underlined nucleotide units designates a 2-deoxynucleotide,or (ii) the 3′-terminal underlined nucleotide designates abeta-D-oxy-LNA nucleotide analogue and the 5′-terminal underlinednucleotide units designates a 2-deoxynucleotide, or (iii) the3′-terminal as well as the 5′-terminal underlined nucleotides designatea beta-D-oxy-LNA nucleotide analogue.

In a further variant, the nucleotide units in the bracket, (T _(x)) or(G _(x)), respectively, is present, and both of the underlinednucleotide units designate a 2-deoxynucleotide.

Although the sequences referred to as SEQ ID NO. 3 and SEQ ID NO. 4 (andmore particular the sequences referred to as SEQ ID NO. 1 and SEQ ID NO.2 (see further below)) are believed to substantially represent the fullfunctionality of the defined LNA oligonucleotides, extension of SEQ IDNO. 3 and SEQ ID NO. 4 with up to five 2-deoxynucleotide units, e.g. 1unit, 2 units, 3 units, 4 units, or even 5 units, is believed to bepossible without detrimental effects on the beneficial properties of thebase sequences, SEQ ID NO. 3 and SEQ ID NO. 4.

This being said, the sequence may be extended at the 3′-terminal end,the 5′-terminal end or at the 3′-terminal end as well as at the5′-terminal end, provided that the total number of 2-deoxynucleotideunits does not exceed 5.

Hence, in one embodiment (which may be combined with the foregoing) theLNA oligonucleotide consists of 15, 16, 17, 18, 19 or 20 nucleotideunits selected from 2-deoxynucleotides and beta-D-oxy-LNA nucleotideanalogues, in particular the LNA oligonucleotide consists of 16nucleotide units selected from 2-deoxynucleotides and beta-D-oxy-LNAnucleotide analogues. In other embodiments (which may be combined withthe foregoing) the LNA oligonucleotide consists of 13, 14, 15, or 16nucleotide units selected from 2-deoxynucleotides and beta-D-oxy-LNAnucleotide analogues, in particular the LNA oligonucleotide consists of14 or 15 nucleotide units selected from 2-deoxynucleotides andbeta-D-oxy-LNA nucleotide analogues.

At least for the sake of convenience in the preparation of the LNAoligonucleotides, it is often preferred that the sequence is extended byone 2-deoxynucleotide unit at the 3′-end, cf., e.g., SEQ ID NO. 1 andSEQ ID NO. 2 below. Most preferable, SEQ ID NO. 3 is extended by anadenosine 2-deoxynucleotide unit at the 3′-end, and SEQ ID NO. 4 isextended by a cytosine 2-deoxynucleotide at the 3′-end.

As mentioned above, subscript “s” designates a phosphorothioate(—O—P(O,S)—O—) link between neighbouring nucleotides/LNA nucleotideanalogues, and subscript “x” designates either a phosphorothioate(—O—P(O,S)—O—) link or a phosphorodiester (—O—P(O)₂—O—) link betweenneighbouring nucleotides/LNA nucleotide analogues. It follows that any2-deoxynucletides by which the sequence is extended may be linked byeither either a phosphorothioate (—O—P(O,S)—O—) link or aphosphorodiester (—O—P(O)₂—O—) link.

It is noted that subsequencec_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T of SEQ ID NO. 3 andsubsequence a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T of SEQ ID NO.4 are indicated as fully phosphorothiolated, cf. subscript “s”. Althoughis it is not currently preferred, it is believed that one, and possiblyalso two, of the phosphorothioate links may be replaced by other links,in particular phosphorodiester links, without severely compromising thestability of the LNA oligonucleotide. Thus, such variants where one ortwo of the phosphorothioate links are replaced by, e.g.,phosphorodiester links also fall within the intended scope of thepresent invention.

In one currently preferred embodiment, however, all nucleotide units inthe sequence are linked by a phosphorothioate group.

One subgroup of particularly interesting LNA oligonucleotides are thoseselected from the group consisting of

(SEQ ID NO. 1)5′-T_(x)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′,(SEQ ID NO. 15)5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T-3′,and (SEQ ID NO. 16)5′-G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)t-3′.

Among those,

(SEQ ID NO. 1)5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′is currently most preferred.

Another subgroup of particularly interesting LNA oligonucleotides arethose selected from the group consisting of

(SEQ ID NO. 2)5′-G_(s)T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A_(s)c-3′,(SEQ ID NO. 17)5′-G_(s)T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A-3′,and (SEQ ID NO. 18)5′-T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)a-3′.

Among those

(SEQ ID NO. 2)5′-G_(s)T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A_(s)c-3′is currently most preferred.

In the present context, the term “nucleoside” is used in its normalmeaning, i.e. it contains a 2-deoxyribose or ribose unit which is bondedthrough its number one carbon atom to one of the nitrogenous basesadenine (A), cytosine (C), thymine (T), uracil (U) or guanine (G).

In a similar way, the term “nucleotide” means a 2-deoxyribose or riboseunit which is bonded through its number one carbon atom to one of thenitrogenous bases adenine (A), cytosine (C), thymine (T), uracil (U) orguanine (G), and which is bonded through its number five carbon atom toan internucleoside phosphate group, or to a terminal group.

The term “nucleic acid” is defined as a molecule formed by covalentlinkage of two or more nucleotides. The terms “nucleic acid” and“polynucleotide” are used interchangeable herein. The term “nucleic acidanalogue” refers to a non-natural nucleic acid binding compound.

The term “LNA monomer” typically refers to a bicyclic nucleosideanalogue, as described in International Patent Application WO 99/14226and subsequent applications, WO 00/56746, WO 00/56748, WO 00/66604, WO00/125248, WO 02/28875, WO 2002/094250 and WO 03/006475 all incorporatedherein by reference.

Beta-D-oxy-LNA is the LNA nucleotide analogue use in the LNAoligonucleotides of the present invention, and the monomer structure(nucleoside) is shown in Scheme 1.

In Scheme 1, Z* and Z indicate the position of a internucleotide linkageto a neighbouring nucleoside or a terminal group (i.e. either a5′-terminal group or a 3′-terminal group).

One particular example of beta-D-oxy-LNA monomer is the thymidine LNAmonomer (LNA nucleoside analogue) (1S,3R, 4R,7S)-7-hydroxy-1-hydroxymethyl-5-methyl-3-(thymin-1yl)-2,5-dioxa-icyclo[2:2:1]heptane,i.e. T-beta-D-oxy-LNA.

The term “oligonucleotide” refers, in the context of the presentinvention, to an oligomer (also called oligo) or nucleic acid polymer(e.g. ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or nucleicacid analogue of those known in the art, preferably Locked Nucleic Acid(LNA), or a mixture thereof. This term includes oligonucleotidescomposed of naturally occurring nucleobases, sugars and internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly or withspecific improved functions. Fully or partly modified or substitutedoligonucleotides are often preferred over native forms because ofseveral desirable properties of such oligonucleotides such as forinstance, the ability to penetrate a cell membrane, good resistance toextra- and intracellular nucleases, high affinity and specificity forthe nucleic acid target. The LNA oligonucleotides of the inventionexhibit the above-mentioned properties.

By the terms “unit” and “nucleotide unit” is understood a monomer, i.e.a 2-deoxynucleotide or a beta-D-oxy-LNA nucleotide analogue.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 andso forth.

The term “a” as used about a nucleoside, a nucleoside analogue, a SEQ IDNO, etc. is intended to mean one or more. In particular, the expression“a component (such as a nucleoside, a nucleoside analogue, a SEQ ID NOor the like) selected from the group consisting of . . . ” is intendedto mean that one or more of the cited components may be selected. Thus,expressions like “a component selected from the group consisting of A, Band C” is intended to include all combinations of A, B and C, i.e. A, B,C, A+B, A+C, B+C and A+B+C.

Throughout this specification, the word “comprise”, or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

Preparation of the LNA Oligonucleotides

The LNA nucleotide analogue building blocks (β-D-oxy-LNA) can beprepared following published procedures and references cited therein,see, e.g., WO 03/095467 A1; D. S. Pedersen, C. Rosenbohm, T. Koch (2002)Preparation of LNA Phosphoramidites, Synthesis 6, 802-808; and WO2004/069991 A2.

The LNA oligonucleotides can be prepared as described in the Examplesand in WO 99/14226, WO 00/56746, WO 00/56748, WO 00/66604, WO 00/125248,WO 02/28875, WO 2002/094250 and WO 03/006475. Thus, the LNAoligonucleotides may be produced using the oligomerisation techniques ofnucleic acid chemistry well-known to a person of ordinary skill in theart of organic chemistry. Generally, standard oligomerisation cycles ofthe phosphoramidite approach (S. L. Beaucage and R. P. Iyer,Tetrahedron, 1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron,1992, 48, 2223) are used, but e.g. H-phosphonate chemistry,phosphotriester chemistry can also be used.

For some monomers, longer coupling time, and/or repeated couplingsand/or use of more concentrated coupling reagents may be necessary orbeneficial.

The phosphoramidites employed couple typically with satisfactory >95%step-wise yields. Oxidation of the phosphorous(III) to phosphorous(V) isnormally done with e.g. iodine/pyridine/H₂O. This yields afterdeprotection the native phosphorodiester internucleoside linkage. In thecase that a phosphorothioate internucleoside linkage is prepared athiolation step is performed by exchanging the normal, e.g.iodine/pyridine/H₂O, oxidation used for synthesis of phosphorodiesterinternucleoside linkages with an oxidation using the ADTT reagent(xanthane hydride (0.01 M in acetonitrile:pyridine 9:1; v/v)). Otherthiolation reagents are also possible to use, such as Beaucage and PADS.The phosphorothioate LNA oligonucleotides were efficiently synthesizedwith stepwise coupling yields >=98%.

Purification of LNA oligonucleotides can be accomplished usingdisposable reversed phase purification cartridges and/or reversed phaseHPLC and/or precipitation from ethanol or butanol. Capillary gelelectrophoresis, reversed phase HPLC, MALDI-MS, and ESI-MS were used toverify the purity of the synthesized LNA oligonucleotides.

Salts

The LNA oligonucleotide can be employed in a variety of pharmaceuticallyacceptable salts. As used herein, the term refers to salts that retainthe desired biological activity of the LNA oligonucleotide and exhibitminimal undesired toxicological effects. Non-limiting examples of suchsalts can be formed with organic amino acid and base addition saltsformed with metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium,and the like, or with a cation formed from ammonia,N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, orethylenediamine; or combinations, e.g., a zinc tannate salt or the like.

Such salts are formed, from the LNA oligonucleotide which possessphosphorodiester group and/or phosphorothioate groups, and are, forexample, salts with suitable bases. These salts include, for example,nontoxic metal salts which are derived from metals of groups Ia, Ib, IIaand IIb of the Periodic System of the elements, in particular suitablealkali metal salts, for example lithium, sodium or potassium salts, oralkaline earth metal salts, for example magnesium or calcium salts. Theyfurthermore include zinc and ammonium salts and also salts which areformed with suitable organic amines, such as unsubstituted orhydroxyl-substituted mono-, di- or tri-alkylamines, in particular mono-,di- or tri-alkylamines, or with quaternary ammonium compounds, forexample with N-methyl-N-ethylamine, diethylamine, triethylamine, mono-,bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine ortris(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxy-loweralkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine ortri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or quaternaryammonium compounds such as tetrabutylammonium salts. Lithium salts,sodium salts, magnesium salts, zinc salts or potassium salts arepreferred, with sodium salts being particularly preferred.

Prodrugs

In one embodiment, the LNA oligonucleotide may be in the form of apro-drug. Oligonucleotides are by virtue negatively charged ions. Due tothe lipophilic nature of cell membranes, the cellular uptake ofoligonucleotides is reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140). In this approach, the LNA oligonucleotides areprepared in a protected manner so that the LNA oligonucleotides areneutral when it is administered. These protection groups are designed insuch a way that they can be removed then the LNA oligonucleotide istaken up be the cells. Examples of such protection groups areS-acetylthioethyl (SATE) or S-pivaloylthioethyl (t-butyl-SATE). Theseprotection groups are nuclease resistant and are selectively removedintracellulary.

Conjugates

A further aspect of the invention relates to a conjugate comprising anLNA oligonucleotide as defined herein at least one non-nucleotide ornon-polynucleotide moiety covalently attached to said LNAoligonucleotide.

In a related aspect of the invention, the LNA oligonucleotide of theinvention is linked to ligands so as to form a conjugate, said ligandsintended to increase the cellular uptake of the conjugate relative tothe antisense oligonucleotides.

In the present context, the term “conjugate” is intended to indicate aheterogenous molecule formed by the covalent attachment of an LNAoligonucleotide as described herein (i.e. an LNA oligonucleotidecomprising a sequence of nucleosides and LNA nucleoside analogues) toone or more non-nucleotide or non-polynucleotide moieties.

Thus, the LNA oligonucleotides may, e.g., be conjugated or form chimerawith non-nucleotide or non-polynucleotide moieties including PeptideNucleic Acids (PNA), proteins (e.g. antibodies for a target protein),macromolecules, low molecular weight drug substances, fatty acid chains,sugar residues, glycoproteins, polymers (e.g. polyethyleneglycol),micelle-forming groups, antibodies, carbohydrates,receptor-binding groups, steroids such as cholesterol, polypeptides,intercalating agents such as an acridine derivative, a long-chainalcohol, a dendrimer, a phospholipid and other lipophilic groups orcombinations thereof, etc., just as the LNA oligonucleotides may bearranged in dimeric or dendritic structures. The LNA oligonucleotides orconjugates of the invention may also be conjugated or further conjugatedto active drug substances, for example, aspirin, ibuprofen, a sulfadrug, an antidiabetic, an antibacterial agent, a chemotherapeutic agentor an antibiotic.

Conjugating in this way may confer advantageous properties with regardto the pharmacokinetic characteristics of the LNA oligonucleotides. Inparticular, conjugating in this way achieves increased cellular uptake.

In one embodiment, an LNA oligonucleotide is linked to ligands so as toform a conjugate, said ligands intended to increase the cellular uptakeof the conjugate relative to the antisense LNA oligonucleotides. Thisconjugation can take place at the terminal positions 5′/3′-OH but theligands may also take place at the sugars and/or the bases. Inparticular, the growth factor to which the antisense LNA oligonucleotidemay be conjugated, may comprise transferrin or folate.Transferrin-polylysine-oligonucleotide complexes orfolate-polylysine-oligonucleotide complexes may be prepared for uptakeby cells expressing high levels of transferrin or folate receptor. Otherexamples of conjugates/ligands are cholesterol moieties, duplexintercalators such as acridine, poly-L-lysine, “end-capping” with one ormore nuclease-resistant linkage groups such as phosphoromonothioate, andthe like.

The preparation of transferrin complexes as carriers of oligonucleotideuptake into cells is described by Wagner et al., Proc. Natl. Acad. Sci.USA 87, 3410-3414 (1990). Cellular delivery of folate-macromoleculeconjugates via folate receptor endocytosis, including delivery of anantisense oligonucleotide, is described by Low et al., U.S. Pat. No.5,108,921. Also see, Leamon et al., Proc. Natl. Acad. Sci. 88, 5572(1991).

Pharmaceutical Composition

A particularly interesting aspect of the invention is directed to apharmaceutical composition comprising an LNA oligonucleotide as definedherein or a conjugate as defined herein, and a pharmaceuticallyacceptable diluent, carrier or adjuvant. The pharmaceutical compositionis preferably suitable for injection, for topical administration, or forintraocular administration (see further below).

Directions for the preparation of pharmaceutical compositions can befound in “Remington: The Science and Practice of Pharmacy” by Alfonso R.Gennaro, and in the following.

Pharmaceutically acceptable diluents, carriers or adjuvants are part ofthe pharmaceutical composition. Capsules, tablets and pills etc. maycontain for example the following compounds: microcrystalline cellulose,gum or gelatin as binders; starch or lactose as excipients; stearates aslubricants; various sweetening or flavouring agents. For capsules thedosage unit may contain a liquid carrier like fatty oils. Likewisecoatings of sugar or enteric agents may be part of the dosage unit. Thepharmaceutical composition may also be emulsions of the activepharmaceutical ingredients (including the LNA oligonucleotide) and alipid forming a micellular emulsion.

An LNA oligonucleotide may be mixed with any material that do not impairthe desired action, or with material that supplement the desired action.These could include other drugs including other oligonucleosidecompounds.

For parenteral, subcutaneous, intradermal or topical administration, theformulation may include a sterile diluent (e.g. water), buffer(s),regulators of tonicity and ionic strength and antibacterials. The activeLNA oligonucleotide may be prepared with carriers that facilitateuptake, protect against degradation or protect against immediateelimination from the body, including implants or microcapsules withcontrolled release properties. For intravenous administration thepreferred carriers are physiological saline (0.9%) or buffered saline(e.g. phosphate buffered saline).

In a preferred embodiment, injections or infusions of the LNAoligonucleotides are given at or near the site of neovascularization.For example, the LNA oligonucleotides of the invention can be deliveredto retinal pigment epithelial cells in the eye. Preferably, the LNAoligonucleotides is administered topically to the eye, e.g. in liquid orgel form to the lower eye lid or conjunctival cul-de-sac, as is withinthe skill in the art (see, e.g., Acheampong A A et al, 2002, DrugMetabol. and Disposition 30: 421-429, the entire disclosure of which isherein incorporated by reference).

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be (a) oral, (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration. Inone embodiment, the active LNA oligonucleotide is administeredintravenous, intraperitonal, orally, topically or as a bolus injectionor administered directly in to the target organ.

It is currently believed that the most appropriate administration formis by intravenous infusions or oral.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, sprays, suppositories, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Compositionsand formulations for oral administration include but are not restrictedto powders or granules, microparticulates, nanoparticulates, suspensionsor solutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Compositions and formulations forparenteral, intrathecal or intraventricular administration may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27).

A particularly preferred parenteral administration route is intraocularadministration. It is understood that intraocular administration of thepresent LNA oligonucleotides can be accomplished by injection or direct(e.g., topical) administration to the eye, as long as the administrationroute allows the LNA oligonucleotides to enter the eye. In addition tothe topical routes of administration to the eye described above,suitable intraocular routes of administration include intravitreal,intraretinal, subretinal, subtenon, peri- and retro-orbital,trans-corneal and trans-scleral administration.

For intraocular administration, the pharmaceutical composition may beadministered topically, for example, by patch or by direct applicationto the eye, or by iontophoresis. Ointments, sprays, or droppable liquidscan be delivered by ocular delivery systems known in the art such asapplicators or eyedroppers. The compositions can be administereddirectly to the surface of the eye or to the interior of the eyelid.Such compositions can include mucomimetics such as hyaluronic acid,chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinylalcohol), preservatives such as sorbic acid, EDTA or benzylchroniumchloride, and the usual quantities of diluents and/or carriers.

The LNA oligonucleotide of the invention may be provided in sustainedrelease compositions, such as those described in, for example, U.S. Pat.Nos. 5, 672,659 and 5,595,760. The use of immediate or sustained releasecompositions depends on the nature of the condition being treated. Ifthe condition consists of an acute or over-acute disorder, treatmentwith an immediate release form will be preferred over a prolongedrelease composition. Alternatively, for certain preventative orlong-term treatments, a sustained release composition may beappropriate.

An LNA oligonucleotide can be injected into the interior of the eye,such as with a needle or other delivery device.

In one embodiment, the pharmaceutical compositions comprise an LNAoligonucleotide of the invention (e.g., 0.1 to 90% by weight), or aphysiologically acceptable salt thereof, mixed with a physiologicallyacceptable carrier medium. Preferred physiologically acceptable carriermedia are water, buffered water, normal saline, 0.4% saline, 0.3%glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For solid compositions, conventional non-toxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

Preferably, an LNA oligonucleotide is included in a unit formulationsuch as in a pharmaceutically acceptable carrier or diluent in an amountsufficient to deliver to a patient a therapeutically effective amountwithout causing serious side effects in the treated patient.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels and suppositories. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In preferred embodiments of the pharmaceutical compositions, the LNAoligonucleotide is formulated in an aqueous carrier, in particular anaqueous carrier comprising a buffer for keeping the pH in the range of4.0-8.5, and having an ionic strength of 20-2000 mM.

The term “aqueous carrier” means that the pharmaceutical composition inquestion is in liquid form, and that the liquid carrier predominantly iscomposed of water, i.e. that at least 80% (w/w), or at least 90% (w/w),or even at least 95% (w/w), of the carrier consists of water. Otherliquid ingredients may also be used, e.g. ethanol, DMSO, ethyleneglycol, etc.

The aqueous carrier preferably comprises saline or a buffer for keepingthe pH in the range of 4.0-8.5. Preferably, the buffer will keep the pHin the range of 5.0-8.0, such as in the range of 6.0-7.5, such asbuffered saline, e.g. phosphate buffered saline (PBS).

The ionic strength/tonicity of the pharmaceutical composition is also ofimportance. Thus, typically, the liquid pharmaceutical composition hasan ionic strength of in the range of 20-2000 mM, such as in the range of50-1500 mM, or in the range of 100-1000 mM.

Combination Drugs

It should be understood that the pharmaceutical composition according tothe invention optionally comprises further antisense compounds,chemotherapeutic agents, anti-inflammatory compounds, antiviralcompounds, cytostatic compounds, anti-angiogenetic compounds,anti-proliferative compounds, pro-apoptotic compounds, signaltransduction modulators, kinase inhibitors and/or immuno-modulatingcompounds. It is currently believed that it is particularly interestingto combine the LNA oligonucleotide with at least one chemotherapeuticagents.

As stated, the pharmaceutical composition of the invention may furthercomprise at least one chemotherapeutic agent. The chemotherapeuticcompound is typically selected from the group consisting ofadrenocorticosteroids, such as prednisone, dexamethasone or decadron;altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol);aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole(arimidex); androgens, such as testosterone; asparaginase (elspar);Avastin; bacillus calmette-gurin; bicalutamide (casodex); biphosphanate;bleomycin (blenoxane); bortezomib; busulfan (myleran); carboplatin(paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran);chlorodeoxyadenosine (2-CDA, cladribine, leustatin); cisplatin(platinol); cyclophosphamid; cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etoposide (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); 2-methoxyestradiol (2ME2, Panzem); mitomycin-C (mutamucin);mitoxantrone (novantrone); octreotide (sandostatin); pentostatin(2-deoxycoformycin, nipent); plicamycin (mithramycin, mithracin);prorocarbazine (matulane); streptozocin; tamoxifin (nolvadex); taxol(paclitaxel); teniposide (vumon, VM-26); Thalidomide; thiotepa;topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoic acid);vinblastine (valban); vincristine (oncovin) and vinorelbine (navelbine).

For the treatment of multiple myeloma, chemotherapeutic agents likemelphalan, cyclophosphamid, prednisone, vincristine, doxorubicin,carmustine, dexamethasone, thalidomide, bortezomib, and biphosphanateare preferred.

For the treatment of renal carcinoma, chemotherapeutic agents likegemcitabine, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine, paclitaxel,carboplatin, ifosfamide, doxorubicin, vinblastine, IFN-alpha, and IL-2are preferred.

In one variant, the present invention provides pharmaceuticalcompositions containing (a) one or more LNA oligonucleotides and (b) oneor more other chemotherapeutic compounds which function by anon-antisense mechanism. When used with the LNA oligonucleotides, suchchemotherapeutic compounds may be used individually (e.g. mithramycinand oligonucleotide), sequentially (e.g. mithramycin and oligonucleotidefor a period of time followed by another agent and oligonucleotide), orin combination with one or more other such chemotherapeutic compounds orin combination with radiotherapy. All chemotherapeutic compounds knownto a person skilled in the art including those explicitly mentionedabove are here incorporated as combination treatments with an LNAoligonucleotide according to the invention.

In one embodiment, the pharmaceutical composition is administered incombination with a taxane compound.

The term “taxane compound” is intended to encompass paclitaxel (Taxol®),paclitaxel derivatives, docetaxel, taxotere, modified taxanes, andtaxoid analogues. Paclitaxel (Taxol®) is a diterpene isolated from thebark of the Western (Pacific) yew, Taxus brevifolia and isrepresentative of a class of therapeutic agents having a taxane ringsystem. Paclitaxel and its analogs have been produced by partialsynthesis from 10-deacetylbaccatin III, a precursor obtained from yewneedles and twigs, and by total synthesis. See Holton, et al., J. Am.Chem. Soc. 116:1597-1601 (1994) and Nicolaou, et al., Nature 367:630(1994). Paclitaxel has demonstrated efficacy in several human tumours inclinical trials. See McGuire, et al., Ann. Int. Med. 111:237-279 (1989);Holmes, et al., J. Natl. Cancer Inst. 83:1797-1805 (1991); Kohn et al.,J. Natl. Cancer Inst. 86:18-24 (1994); and Kohn, et al., AmericanSociety for Clinical Oncology 12 (1993). The modified taxane or taxoidanalogs are those compounds having a taxane ring bearing modified sidechains. A number of these analogs have improved properties, such asgreater water solubility and stability than that of naturally occurringpaclitaxel. These analogs are known to those skilled in the art and aredisclosed, for example, in U.S. Pat. Nos. 5,278,324; 5,272,171;5,254,580; 5,250,683; 5,248,796; and 5,227,400, the disclosures of whichare incorporated herein by reference. Paclitaxel and taxotere can beprepared by the methods in WO 93/18210, EP 0 253 739, EP 0 253 739, andWO 92/09589, the disclosures of which are incorporated herein byreference. In particular embodiments, the taxane compound is paclitaxelor taxotere.

The weight ratio between the taxane compound(s) and the LNAoligonucleotide in said composition is typically in the range of 50:1 to1:25, such as in the range of 25:1 to 1:25, or in the range of 10:1 to1:25, or in the range of 1:1 to 1:25, or in the range of 50:1 to 1:10,or in the range of 1:1 to 1:50, or in the range of 25:1 to 1:10.

In a further embodiment, pharmaceutical compositions of the inventionmay contain one or more LNA oligonucleotides and one or more additionalantisense compounds targeted to a second nucleic acid target. Two ormore combined compounds may be used together or sequentially.

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, antiviral drugs, andimmuno-modulating drugs may also be combined in compositions of theinvention. Two or more combined compounds may be used together orsequentially.

Furthermore, the pharmaceutical compositions comprising the LNAoligonucleotides may be used in combination with radiotherapy, etc.

Medical Treatment

LNA oligonucleotides of the invention are useful for a number oftherapeutic applications as indicated herein. In general, therapeuticmethods of the invention include administration of a therapeuticallyeffective amount of an LNA-modified oligonucleotide to a mammal,particularly a human.

Hence, the present invention also relates to an LNA oligonucleotide asdefined herein or a conjugate as defined herein for use as a medicament.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can also be assessedby measurements of drug in the body of the patient or by surrogatemarkers.

Optimum dosages may vary depending on the relative potency of individualoligonucleotides. Generally, it can be estimated based on EC₅₀s found tobe effective in in vitro and in vivo animal models. In general, dosageis from 0.01 μg to 1 g per kg of body weight, and may be given once ormore daily, weekly, monthly or yearly, or even once every 2 to 10 yearsor by continuous infusion for hours up to several months. The repetitionrates for dosing can be estimated based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state. Itis currently believed that the most relevant doses are 0.01 mg to 100mg, such as 0.1 mg to 40 mg, or 0.5 mg to 10 mg, per kg of body weight.Such doses may be given once daily, but more preferably less frequent,e.g. 1-3 times per week, for a period of 1-4 weeks. Maintenance therapymay be continued, e.g. 1-4 times per month or even less frequent such1-10 times per year.

A person skilled in the art will appreciate that LNA oligonucleotidescan be used to combat HIF-1a linked diseases by many differentprinciples, which thus falls within the spirit of the present invention.

As used herein, the terms “target nucleic acid” encompass DNA encodingthe HIF-1a, RNA (including pre-mRNA and mRNA) transcribed from such DNA,and also cDNA derived from such RNA.

As used herein, the term “gene” means the gene including exons, introns,non-coding 5′ and 3′ regions and regulatory elements and all currentlyknown variants thereof and any further variants, which may beelucidated.

As used herein, the term “LNA oligonucleotide” refers to anoligonucleotide which can induce a desired therapeutic effect in humansthrough for example binding by hydrogen bonding to either a target gene“Chimeraplast” and “TFO”, to the RNA transcript(s) of the target gene“antisense inhibitors”, “siRNA”, “miRNA”, “ribozymes” and “oligozymes”or to the protein(s) encoding by the target gene “aptamer”, “spiegelmer”or “decoy”.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts, which maybe identified.

As used herein, the term “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe present invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

As used herein, the term “targeting” an antisense compound to aparticular target nucleic acid means providing the antisenseoligonucleotide to the cell, animal or human in such a way that theantisense compound are able to bind to and modulate the function of itsintended target.

The LNA oligonucleotides may be designed as siRNA's which are smalldouble stranded RNA molecules that are used by cells to silence specificendogenous or exogenous genes by an as yet poorly understood“antisense-like” mechanism.

The clinical effectiveness of antisense oligonucleotides depends to asignificant extent on their pharmacokinetics e.g. absorption,distribution, cellular uptake, metabolism and excretion. In turn, theseparameters are guided significantly by the underlying chemistry and thesize and three-dimensional structure of the oligonucleotide.

Modulating the pharmacokinetic properties of an LNA oligonucleotideaccording to the invention may further be achieved through attachment ofa variety of different moieties. For instance, the ability ofoligonucleotides to pass the cell membrane may be enhanced by attachingfor instance lipid moieties such as a cholesterol moiety, a thioether,an aliphatic chain, a phospholipid or a polyamine to theoligonucleotide. Likewise, uptake of LNA oligonucleotides into cells maybe enhanced by conjugating moieties to the oligonucleotide thatinteracts with molecules in the membrane, which mediates transport intothe cytoplasm.

The pharmacodynamic properties can according to the invention beenhanced with groups that improve LNA oligonucleotide uptake, enhancebiostability such as enhance LNA oligonucleotide resistance todegradation, and/or increase the specificity and affinity ofoligonucleotides hybridisation characteristics with target sequence e.g.a mRNA sequence.

The pharmaceutical composition according to the invention can be usedfor the treatment of many different diseases. Like cancer cellsproliferating vascular endothelial cells are sensitive todown-regulation of HIF-1a expression. The pharmaceutical compositionaccording to the invention can therefore be used in the treatment ofdiseases characterized by abnormal disease causing angiogenesis.Examples of such diseases are cancers in general and artherosclerosis,psoriasis, diabetic retinopathy, macular degeneration, rheumatoidarthritis, asthma, inflammatory bowel disease, warts, allergicdermatitis and Karposis sarcoma.

Generally stated, one aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to a disease caused byabnormal angiogenesis, comprising administering to the mammal atherapeutically effective amount of an LNA oligonucleotide or aconjugate as defined herein.

Furthermore, the invention also relates to a method of inhibitingangiogenesis comprising the administration of an LNA oligonucleotide asdefined herein or a conjugate as defined herein or a pharmaceuticalcomposition as defined herein.

An interesting aspect of the invention is directed to the use of an LNAoligonucleotide as defined herein or as conjugate as defined herein forthe preparation of a medicament for the treatment of a disease selectedfrom artherosclerosis, psoriasis, diabetic retinopathy, maculardegeneration, rheumatoid arthritis, asthma, inflammatory bowel disease,warts, allergic dermatitis, inflammation, and skin inflammation, orother skin related diseases.

The pharmaceutical composition according to the invention can also beused in the treatment of inflammatory disease, inflammations such asskin inflammations or other skin diseases or disorders, e.g. psoriasisand rheumatoid arthritis.

Similarly, another interesting aspect of the invention is directed to amethod for treating a disease selected from the group consisting ofartherosclerosis, psoriasis, diabetic retinopathy, rheumatoid arthritis,asthma, inflammatory bowel disease, warts, allergic dermatitis,inflammation, and skin inflammation, said method comprisingadministering an LNA oligonucleotide as defined herein or a conjugate asdefined herein or a pharmaceutical composition as defined herein to apatient in need thereof.

Particularly interesting are angiogenic diseases include diabeticretinopathy, macular degeneration, psoriasis, rheumatoid arthritisinflammatory bowel disease, and other inflammatory diseases. Thesediseases are characterized by the destruction of normal tissue by newlyformed blood vessels in the area of neovascularization. For example, inmacular degeneration, the choroid is invaded and destroyed bycapillaries. The angiogenesis-driven destruction of the choroid inmacular degeneration eventually leads to partial or full blindness.

The methods of the invention is preferably employed for treatment orprophylaxis against diseases caused by cancer, particularly fortreatment of cancer as may occur in tissue such as lung, breast, colon,prostate, pancreas, liver, thyroid, kidney, brain, testes, stomach,intestine, bowel, spinal cord, sinuses, bladder, urinary tract orovaries cancer.

Furthermore, the invention described herein encompasses a method ofpreventing or treating cancer comprising a therapeutically effectiveamount of a HIF-1a modulating LNA oligonucleotide, including but notlimited to high doses of the LNA oligonucleotide, to a human in need ofsuch therapy. The invention further encompasses the use of a shortperiod of administration of a HIF-1a modulating LNA oligonucleotide.Normal, non-cancerous cells divide at a frequency characteristic for theparticular cell type. When a cell has been transformed into a cancerousstate, uncontrolled cell proliferation and reduced cell death results,and therefore, promiscuous cell division or cell growth is a hallmark ofa cancerous cell type.

Examples of types of cancer, include, but are not limited to,non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g., acuteleukemia such as acute lymphocytic leukemia, acute myelocytic leukemia,chronic myeloid leukemia, chronic lymphocytic leukemia, multiplemyeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma,bile duct carcinoma, choriocarcinoma, cervical cancer, testicularcancer, lung carcinoma, bladder carcinoma, melanoma, head and neckcancer, brain cancer, cancers of unknown primary site, neoplasms,cancers of the peripheral nervous system, cancers of the central nervoussystem, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, small cell lung carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma), heavychain disease, metastases, or any disease or disorder characterized byuncontrolled or abnormal cell growth.

The term “carcinoma” is intended to indicate a malignant tumor ofepithelial origin. Epithelial tissue covers or lines the body surfacesinside and outside the body. Examples of epithelial tissue are the skinand the mucosa and serosa that line the body cavities and internalorgans, such as intestines, urinary bladder, uterus, etc. Epithelialtissue may also extend into deeper tissue layers to from glands, such asmucus-secreting glands.

The term “sarcoma” is intended to indicate a malignant tumor growingfrom connective tissue, such as cartilage, fat, muscles, tendons andbones.

The term “glioma”, when used herein, is intended to cover a malignanttumor originating from glial cells.

In the use of an LNA oligonucleotide of the invention or as conjugate ofthe invention for the manufacture of a medicament for the treatment ofcancer, said cancer may suitably be in the form of a solid tumor.Furthermore, said cancer is also suitably a carcinoma. The carcinoma istypically selected from the group consisting of malignant melanoma,basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-smallcell lung cancer, renal cell carcinoma, bladder carcinoma, recurrentsuperficial bladder cancer, stomach carcinoma, prostatic carcinoma,pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervicaldysplasia, laryngeal papillomatosis, colon carcinoma, colorectalcarcinoma and carcinoid tumors. More typically, said carcinoma isselected from the group consisting of malignant melanoma, non-small celllung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma.The malignant melanoma is typically selected from the group consistingof superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral melagnoma, amelanotic melanoma and desmoplasticmelanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma istypically in the form selected from the group consisting ofosteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibroushistiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may be a glioma.

The LNA oligonucleotides and conjugates defined herein are also believedto be particularly useful for the treatment of a cancer disease selectedfrom the group consisting of multiple myeloma, renal cancer, cervicalcancer, brain cancer, and breast cancer.

The invention also provides a method for treating cancer, said methodcomprising administering an LNA oligonucleotide as defined herein or aconjugate as defined herein or a pharmaceutical composition as definedherein to a patient in need thereof. In one variant, the cancer is inthe form of a solid tumor. The solid cancer may suitably be a carcinomaor a sarcoma or a glioma, as discussed above.

Accordingly, a further aspect of the invention is directed to the use ofan LNA oligonucleotide as defined herein or as conjugate as definedherein for the manufacture of a medicament for the treatment of cancer,wherein said medicament further comprises a chemotherapeutic agentselected from those defined above under “Combination drugs” Suitably,the further chemotherapeutic agent is selected from taxanes such asTaxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a methodfor treating cancer, said method comprising administering an LNAoligonucleotide as defined herein, or a conjugate as defined herein or apharmaceutical composition as defined herein to a patient in needthereof and further comprising the administration of a a furtherchemotherapeutic agent. Said further administration may be such that thefurther chemotherapeutic agent is conjugated to the LNA oligonucleotideof the invention, is present in the pharmaceutical composition, or isadministered in a separate formulation.

In a preferred embodiment, the present invention provides pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which prevent microtubuledepolymerization and tension forming at the kinetochores of sisterchromatids, but not the attachment of microtubules to the kinetochores.Such chemotherapeutic agents include taxanes as defined above, inparticular Taxol, Paclitaxel and Docetaxel. When used with the LNAoligonucleotides of the invention, such chemotherapeutic agents shouldbe used sequentially initiating with oligonucleotide treatment for aperiod of time which sensitises the target cells to subsequentco-treatment with the chemotherapeutic agent by reducing the level ofHIF-1a protein in tumor cells and proliferating endothelial cells of thetumor vasculature.

In another preferred embodiment, the medical treatment using an LNAoligonucleotide according to the present invention is combined withradiation therapy. When used with the LNA oligonucleotides of theinvention, radiation therapy should be used sequentially initiating witholigonucleotide treatment for a period of time which sensitises thetarget cells to subsequent additional radiotherapy by reducing the levelof HIF-1a protein in tumor cells and proliferating endothelial cells ofthe tumor vasculature.

The LNA oligonucleotides of the present invention can also be utilizedfor as research reagents for diagnostics, therapeutics and prophylaxis.In research, the antisense oligonucleotides may be used to specificallyinhibit the synthesis of HIF-1a genes in cells and experimental animalsthereby facilitating functional analysis of the target or an appraisalof its usefulness as a target for therapeutic intervention. Indiagnostics the antisense oligonucleotides may be used to detect andquantitate HIF-1a expression in cell and tissues by Northern blotting,in-situ hybridisation or similar techniques. For therapeutics, an animalor a human, suspected of having a disease or disorder, which can betreated by modulating the expression of HIF-1a is treated byadministering antisense LNA oligonucleotides in accordance with thisinvention. Further provided are methods of treating an animal particularmouse and rat and treating a human, suspected of having or being proneto a disease or condition, associated with expression of HIF-1a byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense LNA oligonucleotides or conjugates orpharmaceutical compositions of the invention.

A further aspect of the invention is directed to a method of inducingapoptosis comprising the administration of an LNA oligonucleotide asherein, a conjugate as defined herein or a pharmaceutical composition asdefined herein. The induction of apoptosis may be in vitro or in vivo.The induction may be done on a cellular assay or within a tissue sampleor within the living mammal.

A related aspect of the invention is directed method of preventingcellular proliferation comprising the administration of an LNAoligonucleotide as defined herein or a conjugate as defined herein or apharmaceutical composition as defined herein. The prevention ofproliferation may be in vitro or in vivo. The prevention may be done ona cellular assay or within a tissue sample or within the living mammal.

Still further, the invention also relates to a method of treating anangiogenic disease comprising the administration of an LNAoligonucleotide as defined herein or a conjugate as defined herein or apharmaceutical composition as defined herein, such that angiogenesisassociated with the angiogenic disease is inhibited.

In one embodiment, the angiogenic disease comprises a tumor associatedwith a cancer; see also above. The cancer is preferably selected fromthe group consisting of breast cancer, lung cancer, head and neckcancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer,esophagus cancer, gastrointestinal cancer, glioma, liver cancer, tonguecancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer,prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skincancer, lymphoma, and blood cancer. Alternatively, the cancer isselected from the group consisting of multiple myeloma, renal cancer,cervical cancer, colon cancer, brain cancer, and breast cancer.

The angiogenic disease may also be selected from the group consisting ofdiabetic retinopathy, macular degeneration, and inflammatory diseases.Particularly, the angiogenic disease is an inflammatory disease selectedfrom inflammatory bowel disease, psoriasis and rheumatoid arthritis.

Treatment of macular degeneration is believed to be particularlyrelevant with the LNA oligonucleotides of the invention?.

Kits

If the pharmaceutical composition in liquid form is under risk of beingsubjected to conditions which will compromise the stability of the LNAoligonucleotide, it may be preferred to produce the finished productcontaining the LNA oligonucleotide in a solid form, e.g. as a freezedried material, and store the product is such solid form. The productmay then be reconstituted (e.g. dissolved or suspended) in a saline orin a buffered saline ready for use prior to administration.

Hence, the present invention also provides a kit comprising

-   (a) a first component containing an LNA oligonucleotide or a    conjugate as defined hereinabove in solid form, and-   (b) a second component containing saline or a buffer solution (e.g.    buffered saline) adapted for reconstitution (e.g. dissolution or    suspension) of said LNA oligonucleotide.

Preferably said saline or buffered saline has a pH in the range of4.0-8.5, and a molarity of 20-2000 mM. In a preferred embodiment thesaline or buffered saline has a pH of 6.0-8.0 and a molarity of 100-500mM. In a most preferred embodiment the saline or buffered saline has apH of 7.0-8.0 and a molarity of 120-250 mM

For such a kit, the LNA oligonucleotide is preferably selected from thegroup consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 15, SEQ IDNO. 16, SEQ ID NO. 17, and SEQ ID NO. 18. More particular, the LNAoligonucleotide is selected from the group consisting of SEQ ID NO. 1and SEQ ID NO. 2.

The invention is further illustrated in a non-limiting manner by thefollowing examples.

EXPERIMENTALS Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives thereof were preparedfollowing published procedures and references cited therein, see, e.g.WO 03/095467 A1 and D. S. Pedersen, C. Rosenbohm, T. Koch (2002)Preparation of LNA Phosphoramidites, Synthesis 6, 802-808.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized using the phosphoramidite approach onan Expedite 8900/MOSS synthesizer (Multiple Oligonucleotide SynthesisSystem) at 1 μmol or 15 μmol scale. For larger scale synthesis an ÄktaOligo Pilot was used. At the end of the synthesis (DMT-on), theoligonucleotides were cleaved from the solid support using aqueousammonia for 1-2 hours at room temperature, and further deprotected for 4hours at 65° C. The oligonucleotides were purified by reverse phase HPLC(RP-HPLC). After the removal of the DMT-group, the oligonucleotides werecharacterized by AE-HPLC, RP-HPLC, and CGE and the molecular mass wasfurther confirmed by ESI-MS. See below for more details.

Preparation of the LNA-Solid Support:

Preparation of the LNA Succinyl Hemiester

5′-O-Dmt-3′-hydroxy-LNA monomer (500 mg), succinic anhydride (1.2 eq.)and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The reaction wasstirred at room temperature overnight. After extractions with NaH₂PO₄0.1 M pH 5.5 (2×) and brine (1×), the organic layer was further driedwith anhydrous Na₂SO₄ filtered and evaporated. The hemiester derivativewas obtained in 95% yield and was used without any further purification.

Preparation of the LNA-Support

The above prepared hemiester derivative (90 μmol) was dissolved in aminimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixedtogether for 1 min. This pre-activated mixture was combined withLCAA-CPG (500 Å, 80-120 mesh size, 300 mg) in a manual synthesizer andstirred. After 1.5 hours at room temperature, the support was filteredoff and washed with DMF, DCM and MeOH. After drying, the loading wasdetermined to be 57 μmol/g (see Tom Brown, Dorcas J. S. Brown. Modernmachine-aided methods of oligodeoxyribonucleotide synthesis. In: F.Eckstein, editor. Oligonucleotides and Analogues A Practical Approach.Oxford: IRL Press, 1991: 13-14).

Elongation of the Oligonucleotide

The coupling of phosphoramidites (A(bz), G(ibu), 5-methyl-C(bz)) orT-β-cyanoethyl-phosphoramidite) is performed by using a solution of 0.1M of the 5′-O-DMT-protected amidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. Thethiolation is carried out by using xanthane chloride (0.01 M inacetonitrile:pyridine 10%). The rest of the reagents are the onestypically used for oligonucleotide synthesis. The protocol provided bythe supplier was conveniently optimised.

Purification by RP-HPLC:

Column: Xterra RP₁₈ Flow rate: 3 mL/min Buffers: 0.1 M ammonium acetatepH 8 and acetonitrile

ABBREVIATIONS

-   DMT: Dimethoxytrityl-   DCI: 4,5-Dicyanoimidazole-   DMAP: 4-Dimethylaminopyridine-   DCM: Dichloromethane-   DMF: Dimethylformamide-   THF: Tetrahydrofurane-   DIEA: N,N-diisopropylethylamine-   PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate-   Bz: Benzoyl-   Ibu: Isobutyryl

Example 3 Design of the LNA Oligonucleotide

TABLE 1 LNA oligonucleotides SEQ ID NO.5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′1 SEQ ID NO.5′-G_(s)T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A_(s)c-3′2 SEQ ID NO. 5′-(T_(x))G_(x)G_(x)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)(T)-3′3 SEQ ID NO. 5′-(G_(x))T_(x)T_(x)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(x)T_(x)(A)-3′4 SEQ ID NO. 5′-TGGc_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)TGTa-3′5 SEQ ID NO. 5′-TGGcaagcatccTGTa-3′ 6 SEQ ID NO.FAM-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′7 SEQ ID NO.5′-C_(s)G_(s)T_(s)c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)c_(s)g_(s)A_(s)A_(s)T_(s)c-3′8 SEQ ID NO.5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)a_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′9 SEQ ID NO.5′-T_(s)G_(s)A_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)A_(s)G_(s)T_(s)a-3′10 SEQ ID NO. 5′-TGGTg_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGA-3′ 11SEQ ID NO. 5′-TTGCg_(s)g_(s)a_(s)c_(s)t_(s)c_(s)g_(s)g_(s)ATGG-3′ 12 SEQID NO.5′-t_(s)g_(s)g_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)t_(s)g_(s)t_(s)a-3′13 SEQ ID NO.5′-T_(s)T_(s)mC_(s)c_(s)t_(s)a_(s)t_(s)g_(s)c_(s)t_(s)g_(s)t_(s)A_(s)T_(s)mC_(s)c-3′14 SEQ ID NO.5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T-3′15 SEQ ID NO.5′-G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)t-3′16 SEQ ID NO.5′-G_(s)T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A_(s)-3′17 SEQ ID NO.5′-T_(s)T_(s)a_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)a-3′18 SEQ ID NO.5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)t-3′19 SEQ ID NO.FAM-C_(s)G_(s)T_(s)c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)c_(s)g_(s)A_(s)A_(s)T_(s)c-3′20

In Table 1, capital letters designate an β-D-oxy-LNA nucleotide analogue(β-D-oxy-LNA), small letters designate a 2-deoxynucleotide, underlinedesignates either a beta-D-oxy-LNA nucleotide analogue or a2-deoxynucleotide subscript “s” designates a phosphorothioate linkbetween neighbouring nucleotides/LNA nucleotide analogues, and nosubscript between neighbouring nucleotides/LNA nucleotide analoguesdesignates a phosphorodiester link, and subscript “x” designates eithera phosphorothioate link or a phosphorodiester link between neighbouringnucleotides/LNA nucleotide analogues, and nucleotide units in a bracket,e.g. (T _(x)) or (G _(x)), respectively, represent an optional unit. AllLNA-C monomers are 5-methyl-C (^(Me)C).

Measurement of Melting Temperature (T_(m)) of the Compounds:

A 3 μM solution of SEQ ID NO. 1 in 10 mM sodium phosphate/100 mMNaCl/0.1 nM EDTA, pH 7.0 was mixed with its complement DNA/RNA 3 μM in10 mM sodium phosphate/100 mM NaCl/0.1 nM EDTA, pH 7.0 at 90° C. for aminute and allowed to cool to room temperature. The T_(m) of the duplexwas then determined by increasing the temperature 1° C./min. from 25 to95° C. The T_(m) of SEQ ID NO. 1 is shown in Table 2 below:

TABLE 2 Sequence\T_(m) DNA RNA SEQ ID NO. 1 64.2° C. 68.4° C.T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a

Example 4 Stability of LNA Oligonucletides in Human or Rat Plasma

LNA oligonucleotide stability was tested in plasma from human or rats(it could also be mouse, monkey or dog plasma). In 45 μl plasma, 5 μlLNA oligonucleotide is added (a final concentration of 20 μM). The LNAoligonucleotides are incubated in plasma for times ranging from 0 to 96hours at 37° C. (the plasma is tested for nuclease activity up to 96hours and shows no difference in nuclease cleavage-pattern). At theindicated time the sample were snap frozen in liquid nitrogen. 2 μL(equals 40 pmol) LNA oligonucleotide in plasma was diluted by adding 15μL of water and 3 μL 6× loading dye (Invitrogen). As marker a 10 bpladder (In vitrogen 10821-015) is used. To 1 μl ladder 1 μl 6× loadingand 4 μl water is added. The samples are mixed, heated to 65° C. for 10min and loaded to a prerun gel (16% acrylamide, 7 M UREA, 1× TBE, prerunat 50 Watt for 1 h) and run at 50-60 Watt for 2½ hours. Subsequently thegel is stained with 1× SyBR gold (molecular probes) in 1× TBE for 15min. The bands were visualised using a phosphoimager from Biorad. (SeeFIG. 1A in rat plasma & FIG. 1B human and rat plasma.)

LNA oligonucleotide stability was tested in plasma from human (it couldalso be rat, mouse, monkey or dog plasma). A final concentration of 20μM (between 1 or 5 μL) of LNA oligonucleotide was add to a total volumeof 20 μL plasma and incubated for the times ranging from 0 to 24 hours(it could be up to 72 hours—the plasma has been tested for nucleaseactivity up to 72 hours and there is no difference in cleavage-pattern).At the indicated time the sample were stored at −80° C. 1 μL (equal s 20pmol) LNA oligonucleotides in plasma was diluted 10× in water and run ona 16% acrylamide, 7 M UREA gel with a 10 bp ladder (from In vitrogen(cat no. 10821-015)). The gel was run at approximately 40 Watt for 2-3hours before it was stained with lx SyBR gold (molecular probes) in1×TBE for 15 min. The bands were visualised using a phosphoimager fromBiorad. (See FIG. 1)

Example 5 In Vitro Model: Cell Culture

The effect of LNA oligonucleotides on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. Target can be expressedendogenously or by transient or stable transfection of a nucleic acidencoding said nucleic acid.

The expression level of target nucleic acid can be routinely determinedusing, for example, Northern blot analysis, Quantitative PCR,Ribonuclease protection assays. The following cell types are providedfor illustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. When cultured underhypoxia or anoxia, O₂ levels were kept at 1-2% or 0-0.5%, respectively.Cells were routinely passaged 2-3 times weekly.

-   15PC3: The human prostate cancer cell line 15PC3 was kindly donated    by Dr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and    was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax    I+gentamicin.-   PC3: The human prostate cancer cell line PC3 was purchased from ATCC    and was cultured in F12 Coon's with glutamine (Gibco)+10%    FBS+gentamicin.-   518A2: The human melanoma cancer cell line 518A2 was kindly donated    by Dr. B. Jansen, Section of experimental Oncology, Molecular    Pharmacology, Department of Clinical Pharmacology, University of    Vienna and was cultured in DMEM (Sigma)+10% fetal bovine serum    (FBS)+Glutamax I+gentamicin.-   U373: The U373 glioblastoma cells were cultured in EMEM (Sigma)    containing 10% fetal bovine serum plus Glutamax I, NEAA, Sodium    Pyruvate and gentamicin at 37° C., 95% humidity and 5% CO₂.-   HeLa: The cervical carcinoma cell line HeLa was cultured in MEM    (Sigma) containing 10% fetal bovine serum gentamicin at 37° C., 95%    humidity and 5% CO₂.-   MPC-11: The murine multiple myeloma cell line MPC-11 was purchased    from ATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum.-   DU-145: The human prostate cancer cell line DU-145 was purchased    from ATCC and maintained in RPMI with Glutamax+10% FBS.-   RCC-4+/−VHL: The human renal cancer cell line RCC4 stably    transfected with plasmid expressing VHL or empty plasmid was    purchased from ECACC and maintained according to manufacturers    instructions.-   786-0: The human renal cell carcinoma cell line 786-0 was purchased    from ATCC and maintained according to manufacturers instructions-   HUVEC: The human umbilical vein endothelial cell line HUVEC was    purchased from Camcrex and maintained in EGM-2 medium.-   K562: The human chronic myelogenous leukaemia cell line K562 was    purchased from ECACC and maintained in RPMI with Glutamax+10% FBS.    U87MG: The human glioblastoma cell line U87MG was purchased from    ATCC and maintained according to the manufacturers instructions.-   B16: The murine melanoma cell line B16 was purchased from ATCC and    maintained according to the manufacturers instructions.-   LNCap: The human prostate cancer cell line LNCap was purchased from    ATCC and maintained in RPMI with Glutamax+10% FBS

Example 6 In Vitro Model: Treatment with Antisense Oligonucleotide

Cell culturing and transfections: U373 or HeLa cells were seeded in12-well plates at 37° C. (5% CO₂) in D growth media supplemented with10% FBS, Glutamax I and Gentamicin.

When the cells were 60-70% confluent, they were transfected induplicates with different concentrations of oligonucleotides (0.2-100nM) using Lipofectamine 2000 (2.5 -5 μg/ml). Transfections were carriedout essentially as described by Dean et al. (1994, JBC 269:16416-16424).In short, cells were incubated for 10 min. with Lipofectamine in OptiMEMfollowed by addition of oligonucleotide to a total volume of 0.5 mltransfection mix per well. After 4 hours, the transfection mix wasremoved, cells were washed and grown at 37° C. for approximately 20hours (mRNA analysis and protein analysis) during either normoxia orhypoxia in the appropriate growth medium. Cells were then harvested forprotein and RNA analysis.

Example 7 In Vitro Model: Extraction of RNA and cDNA Synthesis

Total RNA Isolation

Total RNA was isolated either using RNeasy mini kit (Qiagen cat. no.74104) or using the Trizol reagent (Life technologies cat. no. 15596).

For total RNA isolation using RNeasy mini kit (Qiagen), cells werewashed with PBS, and Cell Lysis Buffer (RTL, Qiagen) supplemented with1% mercaptoethanol was added directly to the wells. After a few minutes,the samples were processed according to manufacturer's instructions.

Tissue samples were homogenised using a Retsch 300MM homogeniser andtotal RNA was isolated using the Trizol reagent or the RNeasy mini kitas described by the manufacturer.

First Strand Synthesis

First strand synthesis was performed using either OmniScript ReverseTranscriptase kit or M-MLV Reverse transcriptase (essentially describedby manufacturer (Ambion)) according to the manufacturer's instructions(Qiagen). When using OmniScript Reverse Transcriptase 0.5 μg total RNAeach sample, was adjusted to 12 μl and mixed with 0.2 μl poly (dT)₁₂₋₁₈(0.5 μg/μl) (Life Technologies), 2 μl dNTP mix (5 mM each), 2 μl 10× RTbuffer, 0.5 μl RNAguard™ RNase Inhibitor (33 units/ml, Amersham) and 1μl OmniScript Reverse Transcriptase followed by incubation at 37° C. for60 min. and heat inactivation at 93° C. for 5 min.

When first strand synthesis was performed using random decamers andM-MLV-Reverse Transcriptase (essentially as described by manufacturer(Ambion)) 0.25 μg total RNA of each sample was adjusted to 10.8 μl inH₂O. 2 μl decamers and 2 μl dNTP mix (2.5 mM each) was added. Sampleswere heated to 70° C. for 3 min. and cooled immediately in ice water andadded 3.25 μl of a mix containing (2 μl 10× RT buffer; 1 μl M-MLVReverse Transcriptase; 0.25 μl RNAase inhibitor). cDNA is synthesized at42° C. for 60 min followed by heating inactivation step at 95° C. for 10min and finally cooled to 4° C.

Example 8 In Vitro and In Vivo Model: Analysis of OligonucleotideInhibition of HIF-1a Expression by Real-Time PCR

Antisense modulation of HIF-1a expression can be assayed in a variety ofways known in the art. For example, HIF-1a mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), Ribonuclease protection assay (RPA) or real-timePCR. Real-time quantitative PCR is presently preferred. RNA analysis canbe performed on total cellular RNA or mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysisare routine in the art and is taught in, for example, Current Protocolsin Molecular Biology, John Wiley and Sons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available iQ Multi-Color Real Time PCR Detection Systemavailable from BioRAD.

Real-Time Quantitative PCR Analysis of HIF-1a mRNA Levels

Quantitation of mRNA levels was determined by real-time quantitative PCRusing the iQ Multi-Color Real Time PCR Detection System (BioRAD)according to the manufacturers instructions.

Real-time Quantitative PCR is a technique well-known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

Platinum Quantitative PCR SuperMix UDG 2× PCR master mix was obtainedfrom Invitrogen cat# 11730. Primers and TaqMan® probes were obtainedfrom MWG-Biotech AG, Ebersberg, Germany

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA or β-actinmRNA quantity was used as an endogenous control for normalizing anyvariance in sample preparation.

The sample content of human GAPDH mRNA was quantified using the humanGAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystemscat. no. 4310884E) according to the manufacturer's instructions.

For human HIF-1a, the PCR primers were: forward primer:5′-CTCATCCAAGAAGCCCTAACGTGTT-3′ (SEQ ID NO. 21) (final concentration inthe assay; 0.9 μM) reverse primer: 5′-GCTTTCTCTGAGCATTCTGCAAAGC-3′ (SEQID NO. 22) (final concentration in the assay; 0.9 μM) and the PCR probewas: 5′ FAM-CCTCAGGAACTGTAGTTCTTTGACTCAAAGCGACA-TAMRA 3′ (SEQ ID NO. 23)(final concentration in the assay; 0.1 μM).

For cynomolgus HIF-1a, the PCR primers were: I forward primer:5′-GCTTACCATCAGCTATTTGCGTGTG-3′ (final concentration in the assay; 0.9μM) (SEQ ID NO. 24) reverse primer: 5′-GAACCATAACAAAACCATCCAAGGC-3′ (SEQID NO. 25) (final concentration in the assay; 0.9 μM) and the PCR probewas: 5′ FAM-TCATCTTCAATATCCAAATCACCAGCATCCAGAAG-TAMRA 3′ (SEQ ID NO. 26)(final concentration in the assay; 0.1 μM).

For quantification of 18S ribosomal RNA, the TaqMan Eukaryotic 18S rRNAEndogenous Control reagent, (PART# 4310875, Applied Biosystems) was usedaccording to the manufacturers instructions.

For quantification of mouse GAPDH mRNA the following primers and probeswere designed: Sense primer 5′-AAGGCTGTGGGCAAGGTCATC-3′ (SEQ ID NO. 27)(0.3 μM final concentration),

antisense primer 5′-GTCAGATCCACGACGGACACATT-3′ (SEQ ID NO. 28) (0.6 μMfinal concentration),

TaqMan probe 5′-FAM-GAAGCTCACTGGCATGGCATGGCCTTCCGTGTTC-TAMRA-3′ (SEQ IDNO. 29) (0.2 μM final concentration).

Real Time PCR Using Taqman Probes

The cDNA from the first strand synthesis performed as described inexample 6 was diluted 2-20 times, and analyzed by real time quantitativePCR. The primers and probe were mixed with 2×Platinum Quantitative PCRSuperMix UDG (cat. # 11730, Invitrogen) and added to 3.3 μl cDNA to afinal volume of 25 μl. Each sample was analysed in triplicates. Assaying2 fold dilutions of a cDNA that had been prepared on material purifiedfrom a cell line expressing the RNA of interest generated standardcurves for the assays. Sterile H₂O was used instead of cDNA for the notemplate control. PCR program: 50° C. for 2 minutes, 95° C. for 10minutes followed by 40 cycles of 95° C., 15 seconds, 60° C., 1 minutes.

Relative quantities of target mRNA sequence were determined from thecalculated Threshold cycle using the icycler iQ Real-time DetectionSystem software. (See FIG. 2).

SyBR Green Real Time PCR

To determine the relative mouse HIF1α mRNA level cDNA was used inquantitative PCR analysis using an iCycler from BioRad.

To 8 μl of 5-fold diluted cDNA was added 52 μl of a mix containing 29.5μl Platinum qPCR

Supermix-UDG (in-vitrogen), 1030 nM of each primer, 0.57×SYBR Green(Molecular probes) and 11.4 nM Fluorescein (Molecular probes).

Duplicates of 25 μl was used for Q-PCR: 50° C. for 120 sec., 95° C. for120 sec. and 40 cycles [95° C. for 30 sec. and 60° C. for 60 sec.].

HIF1α mRNA expression was normalized to mouse β-actin mRNA which wassimilarly quantified using Q-PCR.

Primers:

mHIF1a: 5′-TGGGACTTTCTTTTACCATGC-3′ (SEQ ID NO. 30) and5′-GGAGTGTTTACGTTTTCCTGAAG-3′ (SEQ ID NO. 31) mβ-actin:5′-CCTTCCTTCTTGGGTATGGAA-3′ (SEQ ID NO. 32) and5′-GCTCAGGAGGAGCAATGATCT-3′ (SEQ ID NO. 33) mVEGF:5′-CACGACAGAAGGAGAGCAGAAGTC-3′ (SEQ ID NO. 34) and5′-GTCGGGGTACTCCTGGAAGATGT-3′ (SEQ ID NO. 35) BCL-2: forward:5′-gccctgtggatgactgagta-3′ (SEQ ID NO. 36) and reverse:5′-cagccaggagaaatcaaacag-3′ (SEQ ID NO. 37)

2-fold dilutions of cDNA synthesised from untreated mouse fibroblasts(Ltk cells) (diluted 5 fold and expressing both HIF1α and β-actin) wasused to prepare standard curves for the assays. Relative quantities ofHIF1α mRNA were determined from the calculated Threshold cycle using theiCycler iQ Real Time Detection System software.

Example 9 In Vitro Analysis: Western Blot Analysis of HIF-1a ProteinLevels

The in vitro effect of HIF-1a LNA oligonucleotides on HIF-1a proteinlevels in transfected cells was determined by Western Blotting.

Cells were harvested and lysed in 50 mM Tris-HCl pH 6.8, 10% glycerol,2.5% SDS, 5 mM DTT and 6 M urea supplemented with protease inhibitorcocktail (Roche). Total protein concentrations were measured using a BCAprotein assay kit (Pierce). 20-100 μg total protein was run on 10-12%Bis-Tris gels in MOPS buffer or on 3-8% Tris Acetate gels and blottedonto a PVDF membranes according to manufacture's instructions(Invitrogen). After overnight incubation in blocking buffer (PBS-Tsupplemented with 5% low fat milk powder), the membranes were incubatedovernight with of an anti-HIF-1a antibody, Bcl-2 antibody VEGF antibodyor antibodies detecting other downstream of HIF-1a. As control ofloading, tubulin or actin were detected using monoclonal antibodies fromNeomarker. Membranes were then incubated with secondary antibodies andHIF-1a were visualized using a chromogenic immunodetection kit(Invitrogen) or a chemiluminescens ECL⁺ detection kit (Amersham). (SeeFIG. 2A and FIG. 2B)

Example 10 In Vitro Analysis: Antisense Inhibition of Human HIF-1aExpression Using Antisense Oligonucleotides and Their Effect on theDownstream Targets VEGFA and MMP-2

The LNA oligonucleotides do also have an effect on the downstreamtargets VEGFA and MMP-2 in media from U373 cells. U373 cells are seededto 0.3×10⁶ cells in T25 flasks (time study) or 0.6×10⁶ cells in T80flasks (48 hours conc. study). U373 cells is placed at 37° C. (5% CO₂)in growth media supplemented with 10% FBS, Glutamax I and Gentamicin.The day after seeding cells were transfected with LNA oligonucleotidesin duplicates or triplicates using different concentrations ofoligonucleotides (0.2-10 nM) using Lipofectamine 2000 (2.5 μg/ml).Transfections were carried out essentially as described by Dean et al.(1994, JBC 269:16416-16424). In short, cells were incubated for 10 min.with Lipofectamine in OptiMEM followed by addition of oligonucleotide.After 4 hours, the transfection mix was removed, cells were washed andgrown at 37° C. for approximately 20 hours (mRNA analysis and proteinanalysis) during normoxia or hypoxia in the appropriate growth medium.Supernatant from cells were harvested at the time indicated. Addition ofprotease inhibitors were added prior to storage at −80° C. Human VEGFAelisa (Cat #DVE-00) and MMP-2 elisa (cat # DMP-200) from RD systems wasused according to manufacturer. Dependent on the time of harvestsupernatant was diluted 5-50 fold prior to measurement. See FIGS. 12A-E.

Example 11 Apoptosis Induction by LNA Oligonucleotides

Culturing of Cells

The glioblastoma cell line U373 (ATCC) was cultured in MEM (Sigma)supplemented with 10% fetal bovine serum, Glutamax I, NEAA, SodiumPyruvate and gentamicin at 37° C., 95% humidity and 5% CO₂. When cellreached 60-70% confluency cells were transfected using Lipofectamine2000 (2.5 μg/ml).

The cervical carcinoma cell line HeLa was cultured in MEM (Sigma)containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and5% CO₂. When cell reached 60-70% confluency cells were transfected usingLipofectamine 2000 (5 μg/ml).

Measurement of Active Caspase 3/7 Activity

U373 cells were seeded to a density of 7000 cells per well in white 96well plate (Nunc 136101) in complete MEM the day prior to transfection.The next day cells were washed once in prewarmed OptiMEM followed byaddition of 72 μl OptiMEM containing 2.5 μg/ml Lipofectamine2000 (Invitrogen). Cells were incubated for 7 min before adding 18 μloligonucleotides diluted in OptiMEM. The final oligonucleotideconcentration ranged from 0.2 nM to 100 nM. After 6 hours of treatment,cells were washed in OptiMEM and 100 μl DMEM containing serum was added.Similar 96 well plates with treated U373 cells were cultured undernormoxia or under Hypoxia/anoxia by placing the 96 well plates inanaerocult bags (Merck) until the time of harvest. Plates wereequilibrated to room temperature for 15 min at the time indicated. 100μl of the highly sensitive Caspase 3/7-Glo™ Reagent (Promega) was addeddirectly to the cells in 96 well and plates were incubated for 1 hoursmin before recording luminescence (luciferase activity) in LuminoskanAscent instrument from Thermo Labsystems after further 1 min lag period.The luciferase activity is measured as Relative Light Units per seconds(RLU/s). The data was processed in the Ascent software 2.4.2. and graphsof fold induction in relative to mock were drawn in excel.

Transfected cells incubated with the caspase 3/7 inhibitor, which blockactive caspase 3/7 activity were used to demonstrate specificity of theapoptotic response. Moreover, Staurosporine, camptothecine or taxolinduced cells served as positive control. (See FIG. 3A and FIG. 3B.)

Annexin V-FITC Flow Cytometry Analysis

1×106 HeLa cells were seeded in T75 flasks one day prior totransfection. On the day of transfection, the cells were washed once in37° C. OptiMEM followed by addition of 7 ml OptiMEM containing 2.5 μg/mlLipofectamine2000 (In vitrogen). Cells were incubated for 7 min beforeadding 1700 μl oligonucleotides diluted in OptiMEM to a finalconcentration of 1-25 nM. Mock transfected cells served as control.After 4 hours of treatment, cells were washed in OptiMEM and 10 mlculture medium was added. Following oligonucleotide treatment cells wereallowed to recover for 24-72 hours before they were harvested byscraping and washed twice in PBS. 2×105 cells were incubated with 5 μlAnnexin V-FITC and 10 μl propidium iodide (PI-10 mg/ml) and incubatedfor 15 min at room temperature in the dark. Incubation of transfectedcells with purified recombinant Annexin V (10 μg) prior to addingAnnexin V-FITC were used to demonstrate specificity and selectivity ofthe staining. Moreover, TRAIL (Apo2L) induced HeLa cells (0.5 μg/ml)were used as positive control.

0.6×106 U373 cells were seeded in T75 flasks one day prior totransfection. On the day of transfection, the cells were washed once in37° C. OptiMEM followed by addition of 7 ml OptiMEM containing 2.5 μg/mlLipofectamine2000 (In vitrogen). Cells were incubated for 7 min beforeadding 1700 μl oligonucleotides diluted in OptiMEM to a finalconcentration of 1-25 nM. Mock transfected cells served as control.After 6 hours of treatment cells were washed in OptiMEM and 10 mlculture medium was added. Following oligonucleotide treatment cells wereallowed to recover for 24-48 hours before they were harvested byscraping and washed twice in PBS. 2×105 cells were incubated with 5 μlAnnexin V-FITC and 10 μl propidium iodide (PI-10 mg/ml) and incubatedfor 15 min at room temperature in the dark. Incubation of transfectedcells with purified recombinant Annexin V (10 μg) prior to addingAnnexin V-FITC were used to demonstrate specificity and selectivity ofthe staining. Moreover, Staurosporine (0.2 μM) induced U373 cells wereused as positive control. (See FIGS. 4A and 4B.)

Example 12 Proliferation Inhibition by LNA Oligonucleotides

Cells were treated according to example 11.

Measurement of Proliferating Viable Cells (MTS Assay)

U373 cells were seeded to a density of 7000 cells per well in clear 96well plate (Scientific Orange no. 1472030100) in DMEM the day prior totransfection. The next day cells were washed once in prewarmed OptiMEMfollowed by addition of 72 μl OptiMEM containing 2.5 μg/mlLipofectamine2000 (Invitrogen). Cells were incubated for 7 min beforeadding 18 μl oligonucleotides diluted in OptiMEM. The finaloligonucleotide concentration ranged from 5 nM to 100 nM. After 6 hoursof treatment, cells were washed in OptiMEM and 100 μl serum containingDMEM was added. Similar 96 well plates with treated U373 cells werecultured under normoxia or under Hypoxia/anoxia by placing the 96 wellplates in anaerocult bags (Merck) until the time of harvest. Viablecells were measured at the times indicated by adding 20 μl thetetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES) (CellTiter 96® AQueous One Solution Cell ProliferationAssay, Promega). Viable cells were measured at 490 nm and 650 nm in aPowerwave (Biotek Instruments).

The inhibition of growth rate ΔOD (490-650 nm)/h were plotted againstthe LNA oligonucleotide concentration relative to mock, which were setto 100%. (See FIG. 5A and FIG. 5B).

Example 13 In-Vivo Uptake and Target Down-Regulation of LNAOligonucleotides

Hairy mice were treated either daily or twice a week (5 times) during a14 days period i.p injection with saline or SEQ ID NO. 1 and differentthiolated versions hereof. SEQ ID NO. 5 is partly thiolated (in the gap)whereas SEQ ID NO. 6 has a phosphodiester backbone. Mice were treatedwith a total dose of 10 mg/kg/14 days, 50 mg/kg/14 days, or 250 mg/kg/14days given either daily or twice weekly.

RNA Purification and cDNA Synthesis from Tissue

Approximately 10 mg tissue was homogenized in 400 μl RTL buffer (Qiagen)supplemented with 1% mercaptoethanol. Total RNA was isolated usingRNeasy mini kit (Qiagen) according to manufacture's instructions.

First strand synthesis was performed using random decamers andM-MLV-Reverse Transcriptase (essentially as described by manufacturer(Ambion)). For each sample, 0.25 μg total RNA was adjusted to 10.8 μl inH₂O. 2 μl decamers and 2 μl dNTP mix (2.5 mM each) was added. Sampleswere heated to 70° C. for 3 min. and cooled immediately in ice water andadded 3.25 μl of a mix containing (2 μl 10× RT buffer; 1 μl M-MLVReverse Transcriptase; 0.25 μl RNAase inhibitor). cDNA is synthesized at42° C. for 60 min followed by heating inactivation step at 95° C. for 10min and finally cooled to 4° C.

Quantitative Real Time PCR Analysis

To determine the relative mouse HIF1α mRNA level in treated anduntreated samples, the generated cDNA was used in quantitative PCRanalysis using an iCycler from BioRad.

To 8 μl of 5-fold diluted cDNA was added 52 μl of a mix containing 29.5μl Platinum qPCR

Supermix-UDG (in-vitrogen), 1030 nM of each primer, 0.57×SYBR Green(Molecular probes) and 11.4 nM Fluorescein (Molecular probes).

Duplicates of 25 μl was used for Q-PCR: 50° C. for 120 sec., 95° C. for120 sec. and 40 cycles [95° C. for 30 sec. and 60° C. for 60 sec.].

HIF1α mRNA expression was normalized to mouse β-actin and/or Gapdh mRNAwhich was similarly quantified using Q-PCR.

mHIF1a: 5′-TGGGACTTTCTTTTACCATGC-3′ (SEQ ID NO. 30) and5′-GGAGTGTTTACGTTTTCCTGAAG-3′ (SEQ ID NO. 31) mβ-actin:5′-CCTTCCTTCTTGGGTATGGAA-3′ (SEQ ID NO. 32) and5′-GCTCAGGAGGAGCAATGATCT-3′ (SEQ ID NO. 33) mVEGF:5′-CACGACAGAAGGAGAGCAGAAGTC-3′ (SEQ ID NO. 34) and5′-GTCGGGGTACTCCTGGAAGATGT-3′ (SEQ ID NO. 35) mGAPDH:5′-AGCCTCGTCCCGTAGACAAAAT-3′ (SEQ ID NO: 38) and5′-GTTGATGGCAACAATCTCCACTTT-3′ (SEQ ID NO: 39) BCL-2: forward:5′-gccctgtggatgactgagta-3′ (SEQ ID NO. 36) and reverse:5′-cagccaggagaaatcaaacag-3′ (SEQ ID NO. 37)

2-fold dilutions of cDNA synthesised from untreated mouse fibroblasts(Ltk cells) (diluted 5 fold and expressing both HIF1α and β-actin) wasused to prepare standard curves for the assays. Relative quantities ofHIF1α mRNA were determined from the calculated Threshold cycle using theicycler iQ Real Time Detection System software.

Extraction of LNA Oligonucleotide from Tissue

Approximately 100 mg tissue was homogenized mechanically in 500 μlExtraction buffer (0.5% Igepal CA-630, 25 mM Tris pH 8.0, 25 mM EDTA,100 mM NaCl containing 1 mg/ml RNAse A) and incubated overnight at 37°C. 500 ml was spiked with reference oligonucleotide and extracted byadding 1 ml phenol-isoamyl-choloroform (25:1:24(v/v/v)). The aqueousphase was transferred to a new tube and extracted again. If necessarythe extract was lyophilized.

IEX HPLC Analysis of Extracted LNA Oligonucleotides

A sample volume of 50 uL was separated over a DNAPac PA-100 (2×250 mm,Dionex) column equipped with a guard column DNAPac PA-100 (2×50 mm,Dionex). The columns were heated to 40° C. The flow rate was 0.25mL/min. and detection wavelength 260 nm. A gradient of the mobile phasesA: TRIS (20 mM), EDTA (1 mM) and sodiumperchlorate (10 mM) pH: 7.6, B:TRIS (20 mM), EDTA (1 mM) and sodiumperchlorate (1M) pH: 7.6, (0-13min., A: 20%, B: 20%; 14-18 min., A: 40%, B: 60%; 22-28 min., A 0%, B:100%; 33-38 min., A: 80%, B: 20%).

FIG. 6A and FIG. 6B show in vivo uptake (in μg per gram tissue) plustarget down-regulation (% inhibition of HIF-1a mRNA expressioncorrelated to β-actin expression relative to saline treated micefollowing i.p. administration of SEQ ID NO. 1 either daily or twice aweek for 14 days (as described above)).

FIG. 6C shows in vivo endogenous kidney target down-regulationadministered ip injections daily in hairy mice for 14 days regimens ofSEQ ID NO. 1.

FIG. 7A shows that SEQ ID NO. 1 is a potent inhibitor in the livermeasured by Q-PCR on HIF-1a expression upon daily administration.

FIG. 7B shows that SEQ ID NO. 1 is also a potent inhibitor in the livermeasured by Q-PCR on HIF-1a expression upon administration twice a week.

FIG. 7C SEQ ID NO. 1 is a potent inhibitor in the kidney measured byQ-PCR on HIF-1a expression upon daily administration.

Example 14 In Vivo Efficacy of SEQ ID NO. 1 in Mice Bearing U373Xenograft Tumours

The effect of oligonucleotide treatment on growth of tumour xenograftson nude mice can be measured using different tumour cell lines. Examplesof such cell lines are human tumour cell lines U87 (glioblastoma), U373(glioblastoma), 15PC3 (prostate cancer), PC3 (prostate cancer), DU145(prostate cancer), LNCap (prostate cancer and murine tumour cell lineB16 (melanoma).

Treatment of subcutaneous tumour xenografts on nude mice using LNAoligonucleotides. Tumour cells were implanted subcutaneously and thenserially passaged by three consecutive Transplantations. Tumourfragments of 1 mm were implanted subcutaneously with a krocar needle inNMRI nude mice. Alternatively, cancer cells typically 10E6 to 10E7 cellssuspended in 300 μL matrigel (BD Bioscience), were subcutaneouslyinjected into the flanks of NMR1: nude mice. Mice were treated byintra-peritoneal injections 5 mg/kg/day. Individual treatment of themice started when tumour volume reached 50 mm³. Treatment with PBS wasinitiated when mean tumour volume of the control (saline treated) groupreached 50 mm³. The experiment was terminated when tumours of any groupreached maximum allowed sizes. The tumour sizes of all mice weremeasured daily by caliper measurements. The effect of treatment wasmeasured as tumour size and tumour growth rate.

In another study using SEQ ID NO. 1, vital tumor pieces from U373 donormice are transplanted onto the fat tissue of the ovaries (day 0) of nudemice. On day four and nine after transplantation mice are treated withLNA oligonucleotide at 50 mg/kg (i.p). Mice are sacrificed 2 days afterthe last dose (day 11) and tumor weight plus staining of tumors withCD-31 ab is performed (See FIGS. 8A and 8C).

FIGS. 8B and 8C show vessel density in U373 tumors from xenografttreated with SEQ ID NO. 1. FIG. 10D shows HIF-1α mRNA expression in U373tumours measured by QPCR.

SEQ ID NO. 1 was dosed at 50 mg/kg twice a week for one week in U373xenograft mice implanted at the ovaries. 2 days following the last doseanimals was sacrificed. Vessel-density was calculated following CD31staining and related to the total area. A statistical significantdifference (P=0.005) was found between the saline group and the micetreated with a scrambled control (SEQ ID NO. 12).

Example 15 Tissue Half-Life and Target Knockdown in Liver and Kidney ofSEQ ID NO. 1

60 NMRI female mice, (app. 25 g) was split in groups of 5 and dosed 30mg/kg SEQ ID NO.1, i.p. (10 mL/kg 2.5 mg/ml) at day 0, 3, 7, 10 and 14.The groups were taken down at day 14. The control groups were dosed with0.9% saline. Tissue samples was taken and prepared in RNA-later.

FIG. 11 shows in vivo uptake (in μg per gram tissue) plus targetdown-regulation (% inhibition of HIF-1a and VEGF mRNA expressioncorrelated to β-actin expression) of mice following 5 i.p. doses of SEQID NO. 1 30mg/kg.

Example 16 Duration of Action and LNA Oligonucleotide Uptake In Vivo

Duration of Action: 20 Balb/cA-nu, female mice, (app. 25 g) PC3,prostate cancer cell line (ECACC#90112714) was split in groups of 5 anddosed 25 mg/kg SEQ ID NO. 7, i.p. (10 mL/kg 2.5 mg/ml) every day fromday 7 to day 13. The groups were taken down one and 5 days after dosing.The control groups were dosed with 0.9% saline. Tissue samples weretaken and prepared in RNA-later. FIG. 10A shows duration of action ofmRNA expression 1 and 5 days post treatment.

LNA oligonucleotide uptake: Following formalin fixation, the tissueswere paraffin embeeded. The tissue were placed in Holt's solution (30 gsaccharose, 1 g acacia gum, 15 mg thymol, distilled water at 100 ml)over night and frozen. Cryosections at 4 my's monted on coated glass andplaced in DAPI solution. The fluorochrome was visualised in flourescencemicroscopy. FIG. 10B shows histological results from tissue from liver,kidney and tumor are from mice treated with a fam-labeled version SEQ IDNO. 1 at 25 mg/kg/day for seven days and sacrificed the 5 days followingthe last treatment. The picture of the skin is from mice treated thesame way, however, sacrificed the day after the last treatment andoverexposed in order to see the weak staining of the basal cells of theskin (the lower blue line). These data suggests the following:

-   Liver: the staining in hepatocytes in mainly located in the    cytoplasm-   Kidney: Very intensive staining of the proximal tubuli and less    staining of the distal tubuli.-   Tumor: Endothelial cell, macrophages are stained (mouse cells).-   Skin: An intense staining of the dermis (endothelial cells and    macrophages) and in the cytoplasma of the basal layer of the    epidermis.

Example 17 LNA Oligonucleotide Uptake and Efficacy In Vivo

At day 0.3×10⁻⁶ cells (PC3 and HT29) were mixed with 300 μl matrixgeland implanted on Balb/cA-nu, female mice, (app. 25 g). On day 7, 10, 13,17 mice were treated by intra-peritoneal injections 5 mg/kg/day witheither saline, a fam labeled version of SEQ ID NO. 1 (SEQ ID NO. 7) or afam labeled version of SEQ ID NO. 8 (SEQ ID NO. 20). Three days (day 20)or 10 days (day 27) after the last dose, the animals were sacrificed.The saline control group was dosed with 0.9% saline. Tissue samples weretaken and prepared in RNA-later until measurement of LNA oligonucleotidecontent by HPLC analysis or analysis of HIF-1a mRNA down-regulation.(see FIGS. 10C-E).

Visualisation of LNA oligonucleotide uptake: Following formalin fixationthe tissues were paraffin embedded. The tissue were placed in Holt'ssolution (30 g saccharose, 1 g acacia gum, 15 mg thymol, distilled wateras 100 ml) over night and frozen. Cryosections at 4 my's monted oncoated glass and placed in DAPI solution. The fluorochrome wasvisualised in flourescence microscopy (data demonstrating the samebiodistribution as in FIG. 10B—data not shown).

Example 18 In Vivo LNA Oligonucleotide Specificity Study of HIF-1α andVEGF

Mismatch study: 15 NMRI female mice, (app. 25 g) were split in groups of5 and dosed 30 mg/kg SEQ ID NO. 1 or SEQ ID NO. 9 i.p. (10 mL/kg, 3.0mg/ml) over 30 sec day 0, 3, 7, 10, 14. The control groups were dosedwith 0.9% saline. The groups were taken down 3-4 hours after lastinjection. Tissue samples were taken and prepared in RNA-later.

FIG. 11 shows in vivo endogenous liver target down-regulation of HIF-1aand VEGF mRNA after 5 doses of 30 mg/kg every 3^(rd) day of SEQ ID NO. 1compared to the one mismatch control SEQ ID NO. 9.

Example 19 In Vivo Potency of a 14 Mer-Version of SEQ ID NO. 1.

NMRI female mice (0.025 kg) were treated by intra-peritoneal injections5 mg/kg/day with SEQ ID NO. 1. Saline animals served as control animalsand were dosed with 0.9% saline. Five animals were sacrificed 1 day or10 days after dosing. Tissue samples were taken and prepared inRNA-later until measurement HIF-1a mRNA expression by QPCR andnormalised to beta-actin as described in M&M.

Example 20 Preparation of the Three-Dimensional Aortic Ring Cultures

Angiogenesis was studied by culturing rings of mouse aorta inthree-dimensional collagen gels with some modifications of the methodoriginally reported for the rat aorta (Masson et al., 2002 Biol PreocedOnline 4(1) p. 24-31). Hairy mice were treated once i.v. with LNAoligonucleotides at a dose ranging from (10 mg/kg to 50 mg/kg). Threedays after dosing the thoracic aortas were removed from the mice,sacrificed by cervical dislocation and immediately transferred to aculture dish containing ice RPMI Medium (Invitrogen) containing 10%Fetal Calf Serum. The peri-aortic fibroadipose tissue was carefullyremoved with fine microdissecting forceps and iridectomy scissors payingspecial attention not to damage the aortic wall. One millimeter longaortic rings (approximately 15 per aorta—a max of 1.5 cm of the aorta)were sectioned and extensively rinsed in 3 consecutive washes of RPMIwith FBS. Ring-shaped explants of mouse aorta were then embedded in 60μL of matrigel (BD biosciences—Matrixgel: 356234) in a well of a 96 wellplate. Following insertion of the aorta another 40 μL of matrigel isadded and left at 37° C. for 10 min to solidify. 100 μL of EGM2(Cambrix) with and without growth factors is added to the wells. As acontrol, aorta rings are additionally covered with EGM2 media containing10 μM Ciplatin. The medium was changed every second day.

Example 21 Quantitative Whole Body Autoradiography Study in Mice afterSingle Intravenous Administration of ³H-Labelled SEQ ID NO. 1

Nine female C57B1/6J (8 weeks Taconic, DK) mice were given 50 mg/kg ofeach test item intravenously in a tail vein 1.5 mCi/kg ³H-SEQ ID NO. 1.

³H-SEQ ID NO. 1 had a specific activity of 155 μCi/mL.

The volume given to each animal was 10 mL/kg of the test formulation.Individual mice were killed at 5 min, 15 min, 1 hour, 4 hours, 24 hours,2 days, 4 days, 7 days and 18 days after administration of each testitem.

For whole body autoradiography, the mice were anaesthetized byisofluran, and then immediately immersed in hexane cooled with dry iceto −80° C., ABR-SOP-0130/04. The frozen carcasses were embedded in a gelof aqueous carboxymethyl cellulose (CMC), frozen in ethanol, cooled withdry ice (−80° C.) and sectioned sagittaly for whole bodyautoradiography, according to the standard method, ABR-SOP-0131/04. Fromeach animal, 20 μm sections were cut at different levels with acryomicrotome (Leica CM 3600) at a temperature of about −20° C. Theobtained sections were caught on tape (Minnesota Mining andManufacturing Co., No. 810) and numbered consecutively with radioactiveink. After being freeze-dried at −20° C. for about 24 hours, selectedsections were covered with a thin layer of talcum powder and put onimaging plates (Fuji, Japan).

Sections were chosen for phosphor imaging to best represent the tissuesand organs of interest. Together with a set of ³H calibration standards,the sections were covered with a thin layer of talcum powder and put onimaging plates. Due to the low energy of ³H, talcum powder was usedinstead of plastic foil in order to protect the image plate. The imagingplates were exposed for 3-7 days at room temperature, enclosed in lighttight cassettes in a lead shielding box to protect from environmentalradiation.

Following exposure the imaging plates were scanned at a pixel size of 50μm using BAS 2500 (Fuji Film Sverige AB, Sweden). The tissues and organsof interest were quantified using AIDA, version 2.43 (Raytest, Germany).

A water-soluble standard test solution of ³H radioactivity was mixedwith whole blood and used for the production of the calibration scale.The standard series consisted of 10 dilutions from 65.44 to 0.30 nCi/mg.For the purpose of quantification, it was assumed that all tissues hadsimilar density and quench characteristics as that of whole blood. Thetissue density was set to 1 g/ml. The limit of quantification wasdefined as the mean concentration value of eight measurements forbackground plus three times the standard deviation value of thesemeasurements.

The various tissues and organs were identified either on theautoradiograms or on the corresponding tissue sections. The term uveaused in this study includes the retinal pigment epithelium representingmelanin containing structures, choroids and sclera of the eye. (seeFIGS. 14A and 14B).

Example 22 Western Blot of HUVEC Cells Transfected with SEQ ID NO. 1

Normal Human Umbilical Vein Endothelial (HUVEC) cells were cultured inCambrix-EGM2 medie were transfected as described in example using 2 and5 nM SEQ ID NO. 1 or 5 nM SEQ ID NO. 8. Following transfection cellswere exposed hypoxia (1% Oxygen) for 16 hours. At harvest cells werewashed in PBS and lysed in a SDS containing lysis buffer (as describedin example). 50 μg was loaded to Tris-Acetate gels and run at 150 V for1 hour. Western blotting was performed as described in example and theblot was incubated in anti-human-HIF-1a (1:500) prior to visualisationby enhanced chemiluminescence. A potent down-regulation by SEQ ID NO. 1is seen, whereas the scrambled control SEQ ID NO. 8 does notdown-regulate HIF-1a expression in HUVEC cells.

Example 23 In Vitro Tubeformation/Capillary-Like Structure FormationAssay

Induction of tubulogenesis was performed using Matrigel (Venetsanakos E,Mirza A, Fanton C et al. Induction of tubulogenesis intelomerase-immortalized human microvascular endothelial cells byglioblastoma cells. Exp Cell Res 2002;273:21-33). Matrigel was thawed onice to prevent premature polymerization; aliquots of 50 μl were platedinto individual wells of 96-well tissue culture plates (Nunc) andallowed to polymerize at 37° C. for at least 30 minutes. TransfectedHUVEC Cells were removed by treatment with trypsin 0.05%-EDTA. The cellswere washed in serum-containing medium then resuspended to 2-x10E5cells/ml. Into each culture well 100-μl transfected or un-transfectedHUVEC cell suspension in culture media with growth factors (VEGF,hFGF-B, R3-IGF-1, hEGF with FBS (2%)) and heparin was added (n=10).Untreated, mock-transfected as well as HUVEC cells transfected with ascrambled control oligo (SEQ ID NO. 8) were used as controls. Dose ofcontrol or test compound was assayed in 6-10 individual wells and theexperiments were performed at least three times. For quantification oftube formation the wells was photographed. (See FIG. 13)

Example 24 FACS Analysis of Uptake in Cells of the Spleen, Bone Marrowand Peripheral Blood

NMRI female mice (0.025 kg) were treated with a fam labelled version ofSEQ ID NO. 1, SEQ ID NO. 7 (50 mg/kg) or an equivalent number ofmolecules of the Fam amidite (at 3 mg/kg) or 0.9% saline. Cells weresacrificed 1 hour post injection and cells from spleen, Peripheral Blood(1 ml to which 1 ml PBS containing 0.1% sodium azide+50 ml heparinsulfate is added—place on ice) or Bone marrow is harvested

Spleen

Place spleen in a metal mesh, and wet with 1 ml R10 (R10 tissue culturemedium containing 10% FCS) containing azide. Push the tissue through themesh and flush through with a total of 4 ml R10+Azide. Remove 0.5 ml oftissue suspension and discard the remainder. The red blood cells arelysed in the suspension by adding 50 ml Red Cell Lysis buffer mix andleave at RT for 10 min. Spin 2000 rpm 10 mins. If necessary to removethe residual red cells repeat this process. Count and block cells.

Spin cells down and resuspend in 1.0 ml FACS buffer containing azide.Assume cell numbers 5×10⁶ cells per spleen for blocking and add 5 μl ofmurine CD16/CD32 per million cells (25 μl Blocking is added).

Peripheral Blood

The red cells are lysed by adding 50 ml of Red Cell Lysis Solution.Cells are spun down and the process is repeated if necessary. Cells arewashed once with PBS, resuspend and count. Non-specific antibody bindingis blocked by adding murine CD16/CD32 at the rate 5 μl per millioncells. Leave at RT for 10 min, then proceed to lineage stains.

Bone Marrow

Cut the bone as close to each end as possible using sterile scissors.Draw up 1 ml of sterile PBS—into 1 ml syringe fitted with a 25G needle.Insert the needle into one end of the bone—usually easiest at theknee—and flush the PBS through the bone. Repeat until the bone is clear.Draw the bone marrow up into the needle several times to break up themarrow. If concerned about the number of red cells a lysis step can beused as above.

Count the cells and block as above. Place 150,000 cells in a sterileeppendorf tube on ice for the Bone Marrow Cultures.

FACS Stains

Lineage stains are performed using specific markers. As described:

Stains 1. CD4 APC, CD8 PE FITC, 7AAD T-cells 2. Gr-1 PE, f4/80 APCneutrophils, macrophages 3. Gr-1 PE, Mac-1APC myelo-monocytic 4. CD34 PElineage APC stem cells 5. B220 APC, CD19 PE B cells 6. CD11b PE, CD11cAPC dendritic cells Isotypes 7. American hamster IgG1 APC CD11c 8. RatIgG2a APC CD4, B220 9. Rat IgG2a PE cd8a, CD19, CD34 10. Rat IgG2b PEGr-1 CD11b

The stains are performed in 96 wells and a total number of 100 μlblocked cells are stained with 100 μl stain mix (either isotype controlsor specific lineage markers). The stains are performed on ice and leftfor 30 min. The cells are spun for 2000 rpm for 2 min. The supernatantis sucked off and the cells are washed with 200 μl FACS buffer andrepeat the centrifugation step. Wash a total of three times. At the endthe cells are resuspended in 200 μl of FACS buffer and add to a FACStube which already contains 200 μl of FACS+5 μl of 7AAD.

FACS analysis was carried out by using Becton Dickinson FACS Calibur(see FIG. 15).

Endothelial cells, granulocytes and CD4+ lymphocytes and macrophages ofperipheral blood and dendritic cells and granulocytes of the bone marrowand granulocytes of the spleen was shown to stain positive forFAM-labeling five days following administration of SEQ ID NO. 7.

Example 25 Hif-1α and Oligonucleotide Content of SEQ ID NO. 1 inCynomolgus Monkey Tissues

In the main toxicity study in cynomolgus monkeys tissues including liverand kidney samples were snap frozen and stored at −70° C. for subsequentanalysis. (see FIGS. 16A and 16B) The monkeys had been treated withintravenous injection of 0, 6, 10 and 40 mg/kg/occasion twice weekly forfour weeks In the groups of animals receiving 0, 10 or 40 mg/kg/occasionsome animals were followed for a recovery period of 4 weeks withouttreatment.

RNA was extracted from samples as described in Example 13 and HIF-1amRNA content was measured as described in Example 8 (see FIG. 16A).Oligonucleotide content was measured as described below (see FIG. 16B).

Sample Preparation: Extraction from Liver and Kidney Tissues

Chemicals/reagents:

Proteinase K(25.1 mg/ml): Sigma P4850.

Phenol-chloroform-isoamyl-alcohol (25:24:1(v/v/v), saturated with 10 mMTris, pH: 8.0, 1 mM EDTA: Sigma P2069

Igepal CA-630: Sigma, I8896

Extraction buffer: 0.5% Igepal CA-630, 25 mM Tris pH 8.0, 25 mM EDTA,100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH)

1 mg/ml of Proteinase K in extraction buffer: Prepared before eachextraction. Tissues (˜100 mg) is weighed off (tissue is kept on dry-icebefore and after weighing). 500 μl extraction buffer containingproteinase K (1 mg/ml) is added. The tissue is homogenized mechanicallyand the homogenate is incubated over night at 37° C.

Reference samples are prepared by dissolving SEQ ID NO. 2 in extractionbuffer at the relevant concentration range. Exactly 100 mg liver tissuefrom un-treated animals is weighed off (kept on dry-ice before and afterweighing). Extraction buffer (with proteinase K, 1 mg/ml) containing thereference material is added to the tissue samples to a total volume of0.5 ml. The tissue is mechanically homogenized and is incubated overnight at 37° C. The detection signal of SEQ ID NO. 2 from these samplesis used to prepare a standard curve covering the lowest and the highestconcentrations found in the treated animals.

Tissue samples are transferred to 2 ml microtubes with screw caps. 1 mlphenol-chloroform-isoamyl-alcohol (25:24:1(v/v/v)) is added followingvigorously shaking for 5 min. Phase separation is achieved bycentrifugation at 4000 RPM for 15 min. The aqueous phase (upper-phase)is transferred to a new tube (compatible with the evaporator) and 500 μlMilli-Q-H₂O is added to the organic phase (residual from the firstextraction). The tubes are stirred vigorously again for 5 min, followingcentrifugation at 4000 RPM for 15 min (SAN039 in room 115). The aqueousphases (water phases from 1. extraction and wash) are pooled andevaporated to dryness (80° C., under nitrogen). The residual isreconstituted in 200 μl Milli-Q-Water following centrifugation at 4000RPM for 15 min. The samples are transferred to HPLC-vials for analysis.

HPLC analysis of oligonucleotide in liver and kidney tissues: Subsequentto the extraction SEQ ID NO. 2 is analysed by ion exchange HPLC:

-   Column:Dionex, DNA pac PA 100: 2×50 mm (guard), 2×250 mm    (analytical)-   Column temp: 42° C.-   Injection vol.: 50 μl-   Wash-solvent: Milli-Q-H₂O-   Purge-solvent: Milli-Q-H₂O-   Detection: UV, 260 nm

Solvents:

-   Buffer A: 1 mM EDTA, 20 mM TRIS-Cl, 10 mM NaClO₄, pH: 7.6 (1 N NaOH)-   Buffer B: 1 mM EDTA, 20 mM TRIS-Cl, 1 M NaClO₄, pH: 7.6 (1 N NaOH)

Example 26 Duration of Action of In Vivo Treatment Using SEQ ID NO.1

Hairy mice were treated with one i.p. injection of 50 mg/kg SEQ IDNO. 1. 5 animals in each group were sacrificed at days 1 and 10 afterdosing (see FIG. 9C) or at days 1, 2, 3, 4, 5 and 10 after dosing (seeFIG. 9B). HIF-1α mRNA expression was analysed by real-time QPCR andnormalised to GAPDH.

Example 27 In Vivo Eye Disease Corneal Model

Mice and anesthesia. BALB/c mice 6-8 weeks of age. Mice wereanesthetized using a mixture of ketamine and xylazine (120 mg/kg bodyweight and 20 mg/kg body weight, respectively).

Mouse model of suture-induced, inflammatory corneal neovascularization.The mouse model of suture-induced inflammatory cornealneovascularization (CNV) was used as previously described by Streilein JW, Bradley D, Sano Y, Sonoda Y. Immunosuppressive properties of tissuesobtained from eyes with experimentally manipulated corneas. Invest.Ophthalmol. Vis. Sci. 1996;37:413-424. Briefly, a 2-mm-diameter cornealtrephine was placed gently on the central cornea of anesthetized micesolely to mark the central corneal area. Three 11-0 sutures were thenplaced intrastromally with two stromal incursions each extending over120° of the corneal circumference. The outer point of suture placementchosen was halfway between the limbus and the line outlined by the 2-mmtrephine; the inner suture point was at the same distance from the 2-mmtrephine line to obtain standardized angiogenic responses. Sutures wereleft in place for 7 days. Mice were euthanized and the cornea withlimbus was excised, and flat-mount double-immunohistochemistry wasperformed. The presence of inflammatory cells in normal corneas andtheir recruitment into corneas 1 week after suture placement wasquantified in hematoxylin and eosin-stained serial sections ofplastic-embedded corneas fixed in 10% paraformaldehyde afterenucleation. In addition, for further characterization of inflammatorycells recruited to the cornea, double immunohistochemistry was performedon corneal whole mounts and frozen sections with the macrophage markersCD11b. The sections was moreover stained for endothelial cells (vesselsby CD31), markers for VEGF, and VEGFR's.

Example 28 The Corneal Micropocket Assay

The corneal micropocket assay was performed as previously described (CaoY, et al. Vascular endothelial growth factor C induces angiogenesis invivo. Proc. Nat. Acad. Sci. U. S. A. 1998;95:14389-14394 ). Briefly,corneal micropockets were created using a modified von Graefe knife, anda micropellet (0.4×0.4 mm) of sucrose aluminum sulfate coated withhydron polymer containing 200 ng of VEGF-A₁₆₄ (R&D) or 200 ng ofrecombinant bfgf (RDI, Flanders, N.J., USA) was implanted into eachpocket. The pellet was positioned 0.6-0.8 mm from the limbus and thesite was covered with antibiotic ointment (erythromycin) and was left inplace for 10 days (n>5-10 mice each). Hemangiogenic and lymphangiogenicresponses were quantified as described above using double immunostainingwith CD31/LYVE-1. The maximal extent of blood versus lymph vesseloutgrowth between subjacent limbus and pellet was gradedsemiquantitatively in four categories for both vessel types: 0, nooutgrowth; 1, outgrowth less than ⅓ of the limbus-pellet distance; 2,outgrowth between ⅓ and ⅔ of the limbus-pellet distance; 3, vesselreaching pellet.

Example 29 In Vivo Psoriasis Model

In vivo Human Skin/SCID Mouse Chimera

Human skin xenografts were orthotopically transplanted onto 7- to8-week-old SCID mice (Taconic, DK) following previously describedprocedures by Wrone-Smith T, Nickoloff B J: Dermal injection ofimmunocytes induces psoriasis. J Clin Invest 1996, 98:1878-1887.Briefly, human skin xenografts measuring 1.5×1.5×0.5 cm were sutured tothe flank of SCID mice with absorbable 5-0 Vicryl Rapide suture(Ethicon, Somerville, N.J.) and covered with Xeroform dressings (KendallCo., Mansfield, Mass.). Dressings were removed 1 week later and animalsmaintained pathogen-free throughout the study. The mice were treatedwith SEW ID NO. 1 and SEQ ID NO. 7 twice a week at 50 mg/kg one-threeweeks after transplantation. Human skin/SCID mouse chimeras were killedfollowing 2-3 weeks of treatment and 4-mm punch biopsies (Baker's BiopsyPunch, Cummins Derm, Miami, Fla.) were obtained from each xenograft.Biopsies were fixed in neutral-buffered formalin for paraffin embeddingand/or mounted on gum tragacanth (Sigma Chemical Co., St. Louis, Mo.),snap-frozen in liquid nitrogen-chilled isopentane, and stored at −80° C.

Immunostaining

Cryostat sections of skin were stained for relevant marker includingendotheial cells (CD31/CD34), macrophages (cd11b) VEGF, VEGFR or HIF-1a.The sections were counter-stained with hematoxylin and eosin (asdescribed previously). All slides were examined and photographed.

1. A method for treating cancer, said method comprising administering atherapeutically effective amount of an LNA oligonucleotide consisting ofthe sequence:5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′(SEQ ID NO. 1) wherein capital letters designate a beta-D-oxy-LNAnucleotide analogue, lower case letters designate a 2-deoxynucleotide,and subscript “s” designates a phosphorothioate link between neighboringnucleotides/LNA nucleotide analogues to a patient in need thereof. 2.The method according to claim 1, wherein said cancer is a solid tumor.3. The method according to claim 1, wherein the cancer is selected fromthe group consisting of multiple myeloma, renal cancer, cervical cancer,colon cancer, brain cancer, and breast cancer.
 4. A method of inducingapoptosis in a cell comprising contacting the cell with an effectiveamount of an LNA oligonucleotide consisting of the sequence:5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′(SEQ ID NO. 1) wherein capital letters designate a beta-D-oxy-LNAnucleotide analogue, lower case letters designate a 2-deoxynucleotide,and subscript “s” designates a phosphorothioate link between neighboringnucleotides/LNA nucleotide analogues.
 5. A method of preventingproliferation of a cell comprising contacting the cell with an effectiveamount of an LNA oligonucleotide consisting of the sequence:5′-T_(s)G_(s)G_(s)c_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)T_(s)G_(s)T_(s)a-3′(SEQ ID NO. 1) wherein capital letters designate a beta-D-oxy-LNAnucleotide analogue, lower case letters designate a 2-deoxynucleotide,and subscript “s” designates a phosphorothioate link between neighboringnucleotides/LNA nucleotide analogues.